Polynucleotide encoding adipocyte complement related protein homolog zacrp2

ABSTRACT

The present invention relates to polynucleotide and polypeptide molecules for zacrp2, a novel member of the family of proteins bearing a collagen-like domain and a C1q domain. The polypeptides and polynucleotides encoding them, are involved in dimerization or oligomerization and may be used in the study thereof. The present invention also includes antibodies to the zacrp2 polypeptides.

REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 09/552,204, filed Apr. 19, 2000, now U.S. Pat. No. 6,620,909 B1,which claims the benefit of U.S. Patent Application Ser. No. 60/130,207,filed Apr. 20, 1999, both of which are herein incorporated by reference.

BACKGROUND OF THE INVENTION

Energy balance (involving energy metabolism, nutritional state, lipidstorage and the like) is an important criteria for health. This energyhomeostasis involves food intake and metabolism of carbohydrates andlipids to generate energy necessary for voluntary and involuntaryfunctions. Metabolism of proteins can lead to energy generation, butpreferably leads to muscle formation or repair. Among otherconsequences, a lack of energy homeostasis lead to over or underformation of adipose tissue.

Formation and storage of fat is insulin-modulated. For example, insulinstimulates the transport of glucose into cells, where it is metabolizedinto α-glycerophosphate which is used in the esterification of fattyacids to permit storage thereof as triglycerides. In addition,adipocytes (fat cells) express a specific transport protein thatenhances the transfer of free fatty acids into adipocytes.

Adipocytes also secrete several proteins believed to modulatehomeostatic control of glucose and lipid metabolism. These additionaladipocyte-secreted proteins include adipsin, complement factors C3 andB, tumor necrosis factor α, the ob gene product and Acrp30. Evidencealso exists suggesting the existence of an insulin-regulated secretorypathway in adipocytes. Scherer et al., J. Biol. Chem. 270(45): 26746-9,1995. Over or under secretion of these moieties, impacted in part byover or under formation of adipose tissue, can lead to pathologicalconditions associated directly or indirectly with obesity or anorexia.

Acrp30 is a 247 amino acid polypeptide that is expressed exclusively byadipocytes. The Acrp30 polypeptide is composed of a amino-terminalsignal sequence, a 27 amino acid stretch of no known homology, 22perfect Gly-Xaa-Pro or imperfect Gly-Xaa-Xaa collagen repeats and acarboxy terminal globular domain. See, Scherer et al. as described aboveand International Patent Application No. WO 96/39429. Acrp30, anabundant human serum protein regulated by insulin, shares structuralsimilarity, particularly in the carboxy-terminal globular domain, tocomplement factor C1q and to a summer serum protein of hibernatingSiberian chipmunks (Hib27). Expression of Acrp30 is induced over100-fold during adipocyte differentiation. Acrp30 is suggested for usein modulating energy balance and in identifying adipocytes in testsamples.

Another secreted protein that appears to be exclusively produced inadipocytes is apM1, described, for example, in Maeda et al., Biochem.Biophys. Res. Comm. 221: 286-9, 1996. A 4517 bp clone had a 244 aminoacid open reading frame and a long 3′ untranslated region. The proteinincluded a signal sequence, an amino-terminal non-collagenous sequence,22 collagen repeats (Gly-XAA-Pro or Gly-Xaa-Xaa), and a carboxy-terminalregion with homology to collagen X, collagen VIII and complement proteinC1q.

Complement factor C1q consists of six copies of three relatedpolypeptides (A, B and C chains), with each polypeptide being about 225amino acids long with a near amino-terminal collagen domain and acarboxy-terminal globular region. Six triple helical regions are formedby the collagen domains of the six A, six B and six C chains, forming acentral region and six stalks. A globular head portion is formed byassociation of the globular carboxy terminal domain of an A, a B and a Cchain. C1q is therefore composed of six globular heads linked via sixcollagen-like stalks to a central fibril region. Sellar et al., Biochem.J. 274: 481-90, 1991. This configuration is often referred to as abouquet of flowers. Acrp30 has a similar bouquet structure formed from asingle type of polypeptide chain.

C1q has been found to stimulate defense mechanisms as well as triggerthe generation of toxic oxygen species that can cause tissue damage(Tenner, Behring Inst. Mitt. 93:241-53, 1993). C1q binding sites arefound on platelets. Additionally complement and C1q play a role ininflammation. The complement activation is initiated by binding of C1qto immunoglobulins

Inhibitors of C1q and the complement pathway would be useful foranti-inflammatory applications, inhibition of complement activation andthrombotic activity.

The present invention provides such polypeptides for these and otheruses that should be apparent to those skilled in the art from theteachings herein.

SUMMARY OF THE INVENTION

Within one aspect the invention provides an isolated polypeptidecomprising a sequence of amino acid residues that is at least 75%identical in amino acid sequence to residues 40-285 of SEQ ID NO:2,wherein the sequence comprises: Gly-Xaa-Xaa or Gly-Xaa-Pro repeatsforming a collagen domain, wherein Xaa is any amino acid; and acarboxyl-terminal C1q domain comprises 10 beta strands. Within oneembodiment the polypeptide that is at least 90% identical in amino acidsequence to residues 16-285 of SEQ ID NO:2. Within another embodimentthe collagen domain consists of 24 Gly-Xaa-Xaa repeats and 10Gly-Xaa-Pro repeats. Within another embodiment the carboxyl-terminal C1qdomain comprises the sequence of SEQ ID NO:5. Within another embodimentthe carboxy-terminal C1q domain comprises amino acid residues 151-155,172-174, 180-183, 187-190, 193-205, 208-214, 220-227, 229-241, 246-251and 269-274 of SEQ ID NO:2. Within another embodiment any differencesbetween said polypeptide and SEQ ID NO:2 are due to conservative aminoacid substitutions. Within another embodiment the polypeptidespecifically binds with an antibody that specifically binds with apolypeptide consisting of the amino acid sequence of SEQ ID NO:2. Withina further embodiment the polypeptide comprises residues 16-285 of SEQ IDNO:2. Within another embodiment the polypeptide is covalently linked atthe amino or carboxyl terminus to a moiety selected from the groupconsisting of affinity tags, toxins, radionucleotides, enzymes andfluorophores. Within yet another embodiment the collagen domain consistsof amino acid residues 41-141 of SEQ ID NO:2. Within another embodimentthe carboxy-terminal C1q domain consists of amino acid residues 142-285of SEQ ID NO:2.

The invention also provides an isolated polypeptide selected from thegroup consisting of: a) a polypeptide consisting of a sequence of aminoacid residues that is 75% identical in amino acid sequence to amino acidresidue 40 to amino acid residue 141 of SEQ ID NO:2; b) a polypeptideconsisting of a sequence of amino acid residues that is 75% identical inamino acid sequence to amino acid residue 142 to amino acid residue 285of SEQ ID NO:2; and c) a polypeptide consisting of a sequence of aminoacid residues that is 75% identical in amino acid sequence to amino acidresidue 40 to 285 of SEQ ID NO:2.

Within another aspect, the invention provides a fusion proteincomprising a first portion and a second portion joined by a peptidebond, the first portion consisting of a polypeptide selected from thegroup consisting of: a) a polypeptide comprising a sequence of aminoacid residues that is at least 75% identical in amino acid sequence toamino acid residue 16 to amino acid residue 285 of SEQ ID NO:2; b) apolypeptide comprising a sequence of amino acid residues as shown in SEQID NO:2 from amino acid residue 1 to amino acid residue 281; c) apolypeptide comprising a sequence of amino acid residues as shown in SEQID NO:2 from amino acid residue 16 to amino acid residue 285; d) aportion of the zacrp2 polypeptide as shown in SEQ ID NO:2, comprisingthe collagen-like domain or a portion of the collagen-like domaincapable of dimerization or oligomerization; e) a portion of the zacrp2polypeptide as shown in SEQ ID NO:2, comprising the C1q domain or anactive portion of the C1q domain; or f) a portion of the zacrp2polypeptide as shown in SEQ ID NO:2 comprising of the collagen-likedomain and the C1q domain; and the second portion comprising anotherpolypeptide. Within one embodiment the first portion is selected fromthe group consisting of: a) a polypeptide consisting of the sequence ofamino acid residue 40 to amino acid residue 141 of SEQ ID NO:2; b) apolypeptide consisting of the sequence of amino acid residue 142 toamino acid residue 285 of SEQ ID NO:2; c) a polypeptide consisting ofthe sequence of amino acid residue 40 to 285 of SEQ ID NO:2.

Within another aspect, the invention provides a polypeptide as describedabove; in combination with a pharmaceutically acceptable vehicle.

Within another aspect is provided an antibody or antibody fragment thatspecifically binds to a polypeptide as described above. Within oneembodiment the antibody is selected from the group consisting of: a)polyclonal antibody; b) murine monoclonal antibody; c) humanizedantibody derived from b); and d) human monoclonal antibody. Withinanother embodiment the antibody fragment is selected from the groupconsisting of F(ab′), F(ab), Fab′, Fab, Fv, scFv, and minimalrecognition unit. Within another embodiment is provided an anti-idiotypeantibody that specifically binds to the antibody described above.

Within another aspect, the invention provides an isolated polynucleotideselected from the group consisting of: a) a polynucleotide encoding apolypeptide comprising a sequence of amino acid residues that is atleast 75% identical in amino acid sequence to residues 40-285 of SEQ IDNO:2, wherein the sequence comprises: Gly-Xaa-Xaa or Gly-Xaa-Pro repeatsforming a collagen domain, wherein Xaa is any amino acid; and acarboxyl-terminal C1q domain comprising 10 beta strands. Within oneembodiment the polypeptide is at least 90% identical in amino acidsequence to residues 16-285 of SEQ ID NO:2. Within another embodimentthe collagen domain consists of 24 Gly-Xaa-Xaa repeats and 10Gly-Xaa-Pro repeats. Within yet another embodiment the carboxyl-terminalC1q domain comprises the sequence of SEQ ID NO:5. Within anotherembodiment the carboxy-terminal C1q domain comprises amino acid residues151-155, 172-174, 180-183, 187-190, 193-205, 208-214, 220-227, 229-241,246-251 and 269-274 of SEQ ID NO:2. Within another embodiment anydifferences between said polypeptide and SEQ ID NO:2 are due toconservative amino acid substitutions. Within another embodiment thepolynucleotide encodes a polypeptide that specifically binds with anantibody that specifically binds with a polypeptide consisting of theamino acid sequence of SEQ ID NO:2. Within still another embodiment thepolynucleotide encodes a polypeptide that comprises residues 16-285 ofSEQ ID NO:2. Within another embodiment the collagen domain consists ofamino acid residues 41-141 of SEQ ID NO:2. Within yet another embodimentthe carboxy-terminal C1q domain consists of amino acid residues 142-285of SEQ ID NO:2.

Also provided is an isolated polynucleotide selected from the groupconsisting of: a) a sequence of nucleotides from nucleotide 1 tonucleotide 1161 of SEQ ID NO:1; b) a sequence of nucleotides fromnucleotide 133 to nucleotide 987 of SEQ ID NO:1; c) a sequence ofnucleotides from nucleotide 178 to nucleotide 987 of SEQ ID NO:1; d) asequence of nucleotides from nucleotide 250 to nucleotide 987 of SEQ IDNO:1; e) a sequence of nucleotides from nucleotide 556 to nucleotide 987of SEQ ID NO:1; f) a sequence of nucleotides from nucleotide 133 tonucleotide 555 of SEQ ID NO:1; g) a sequence of nucleotides fromnucleotide 178 to nucleotide 555 of SEQ ID NO:1; h) a sequence ofnucleotides from nucleotide 250 to nucleotide 555 of SEQ ID NO:1; i) apolynucleotide encoding a polypeptide, the polypeptide consisting of asequence of amino acid residues that is at least 75% identical to apolypeptide consisting of the amino acid sequence of residues 40 to 141of SEQ ID NO:2; j) a polynucleotide encoding a polypeptide, thepolypeptide consisting of a sequence of amino acid residues that is atleast 75% identical to a polypeptide consisting of the amino acidsequence of residues 142 to 285 of SEQ ID NO:2; k) a polynucleotideencoding a polypeptide, the polypeptide consisting of a sequence ofamino acid residues that is at least 75% identical to a polypeptideconsisting of the amino acid sequence of residues 40 to 285 of SEQ IDNO:2; l) a polynucleotide encoding a polypeptide consisting of asequence of amino acid residues that is at least 75% identical to apolypeptide consisting of the amino acid sequence of residues 16 to 141of SEQ ID NO:2; m) a polynucleotide that remains hybridized followingstringent wash conditions to a polynucleotide consisting of thenucleotide sequence of SEQ ID NO:1, or the complement of SEQ ID NO:1; n)nucleotide sequences complementary to a), b), c), d), e), f), g), h),i), j), k), l) or m) and o) degenerate nucleotide sequences of i), j),k) or l).

The invention further provides an isolated polynucleotide encoding afusion protein comprises a first portion and a second portion joined bya peptide bond, the first portion is selected from the group consistingof: a) a polypeptide comprising a sequence of amino acid residues thatis at least 75% identical in amino acid sequence to amino acid residues40 to 285 of SEQ ID NO:2; b) a polypeptide comprising the sequence ofamino acid residues 1 to 285 of SEQ ID NO:2; c) a polypeptide comprisingthe sequence of amino acid residues 16 to 285 of SEQ ID NO:2; d) aportion of a polypeptide of SEQ ID NO:2 comprising the collagen-likedomain or a portion of the collagen-like domain capable of dimerizationor oligomerization; e) a portion of the polypeptide of SEQ ID NO:2containing the C1q domain; or f) a portion of the polypeptide of SEQ IDNO:2 including the collagen-like domain and the C1q domain; and thesecond portion comprising another polypeptide.

Also provided is an isolated polynucleotide consisting of the sequenceof nucleotide 1 to nucleotide 855 of SEQ ID NO:10.

Within another aspect, the invention provides an expression vectorcomprising the following operably linked elements: a transcriptionpromoter; a DNA segment encoding a polypeptide as described above; and atranscription terminator. Within one embodiment the DNA segment encodesa polypeptide that is at least 90% identical in amino acid sequence toresidues 16-285 of SEQ ID NO:2. Within another embodiment the collagendomain consists of 24 Gly-Xaa-Xaa repeats and 10 Gly-Xaa-Pro repeats.Within another embodiment the carboxyl-terminal C1q domain comprises thesequence of SEQ ID NO:5. Within another embodiment the carboxy-terminalC1q domain comprises amino acid residues 151-155, 172-174, 180-183,187-190, 193-205, 208-214, 220-227, 229-241, 246-251 and 269-274 of SEQID NO:2. Within another embodiment any differences between saidpolypeptide and SEQ ID NO:2 are due to conservative amino acidsubstitutions. Within another embodiment the collagen domain consists ofamino acid residues 41-141 of SEQ ID NO:2. Within another embodiment thecarboxy-terminal C1q domain consists of amino acid residues 142-285 ofSEQ ID NO:2. Within yet another embodiment the polypeptide specificallybinds with an antibody that specifically binds with a polypeptideconsisting of the amino acid sequence of SEQ ID NO:2. Within yet anotherembodiment the DNA segment encodes a polypeptide comprising residues16-285 of SEQ ID NO:2. Within a further embodiment the DNA segmentencodes a polypeptide covalently linked at the amino or carboxylterminus to an affinity tag. Within another embodiment the DNA segmentfurther encodes a secretory signal sequence operably linked to thepolypeptide. Within a related embodiment the secretory signal sequencecomprises residues 1-15 of SEQ ID NO:2.

Within another aspect, the invention provides a cultured cell into whichhas been introduced an expression vector as described above, wherein thecell expresses the polypeptide encoded by the DNA segment.

Within still another aspect, the invention a method of producing apolypeptide comprising: culturing a cell into which has been introducedan expression vector as described above; whereby the cell expresses thepolypeptide encoded by the DNA segment; and recovering the expressedpolypeptide.

BRIEF DESCRIPTION OF THE DRAWINGS

The FIGURE illustrates a multiple alignment of and zacrp2 polypeptide ofthe present invention and human ACRP30 (ACR3) (SEQ ID NO:3, Maeda etal., Biochem. Biophys. Res. Commun. 221:286-9, 1996) and human C1q C(SEQ ID NO:4, Sellar et al., Biochem J. 274:481-90, 1991 and Reid,Biochem J. 179:361-71, 1979). The multiple alignment performed using aClustalx multiple alignment tool with the default settings: BlosumSeries Weight Matricies, Gap Opening penalty:10.0, Gap Extensionpenalty:0.05. Multiple alignments were further hand tuned beforecomputing percent identity.

DETAILED DESCRIPTION OF THE INVENTION

Prior to setting forth the invention in detail, it may be helpful to theunderstanding thereof to define the following terms.

The term “affinity tag” is used herein to denote a peptide segment thatcan be attached to a polypeptide to provide for purification ordetection of the polypeptide or provide sites for attachment of thepolypeptide to a substrate. In principal, any peptide or protein forwhich an antibody or other specific binding agent is available can beused as an affinity tag. Affinity tags include a poly-histidine tract,protein A (Nilsson et al., EMBO J. 4:1075, 1985; Nilsson et al., MethodsEnzymol. 198:3, 1991), glutathione S transferase (Smith and Johnson,Gene 67:31, 1988), substance P, Flag™ peptide (Hopp et al.,Biotechnology 6:1204-10, 1988; available from Eastman Kodak Co., NewHaven, Conn.), streptavidin binding peptide, or other antigenic epitopeor binding domain. See, in general Ford et al., Protein Expression andPurification 2: 95-107, 1991. DNAs encoding affinity tags are availablefrom commercial suppliers (e.g., Pharmacia Biotech, Piscataway, N.J.).

The term “allelic variant” denotes any of two or more alternative formsof a gene occupying the same chromosomal locus. Allelic variation arisesnaturally through mutation, and may result in phenotypic polymorphismwithin populations. Gene mutations can be silent (no change in theencoded polypeptide) or may encode polypeptides having altered aminoacid sequence. The term allelic variant is also used herein to denote aprotein encoded by an allelic variant of a gene.

The terms “amino-terminal” and “carboxyl-terminal” are used herein todenote positions within polypeptides and proteins. Where the contextallows, these terms are used with reference to a particular sequence orportion of a polypeptide or protein to denote proximity or relativeposition. For example, a certain sequence positioned carboxyl-terminalto a reference sequence within a protein is located proximal to thecarboxyl terminus of the reference sequence, but is not necessarily atthe carboxyl terminus of the complete protein.

The term “complement/anti-complement pair” denotes non-identicalmoieties that form a non-covalently associated, stable pair underappropriate conditions. For instance, biotin and avidin (orstreptavidin) are prototypical members of a complement/anti-complementpair. Other exemplary complement/anti-complement pairs includereceptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs,sense/antisense polynucleotide pairs, and the like. Where subsequentdissociation of the complement/anti-complement pair is desirable, thecomplement/anti-complement pair preferably has a binding affinity of<10⁹ M⁻¹.

The term “complements of a polynucleotide molecule” is a polynucleotidemolecule having a complementary base sequence and reverse orientation ascompared to a reference sequence. For example, the sequence 5′ ATGCACGGG3′ is complementary to 5′ CCCGTGCAT 3′.

The term “contig” denotes a polynucleotide that has a contiguous stretchof identical or complementary sequence to another polynucleotide.Contiguous sequences are said to “overlap” a given stretch ofpolynucleotide sequence either in their entirety or along a partialstretch of the polynucleotide. For example, representative contigs tothe polynucleotide sequence 5′-ATGGCTTAGCTT-3′ (SEQ ID NO:21) are5′-TAGCTTgagtct-3′ (SEQ ID NO:22) and 3′-gtcgacTACCGA-5′ (SEQ ID NO:23).

The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons (as compared toa reference polynucleotide molecule that encodes a polypeptide).Degenerate codons contain different triplets of nucleotides, but encodethe same amino acid residue (i.e., GAU and GAC triplets each encodeAsp).

The term “expression vector” denotes a DNA molecule, linear or circular,that comprises a segment encoding a polypeptide of interest operablylinked to additional segments that provide for its transcription. Suchadditional segments may include promoter and terminator sequences, andmay optionally include one or more origins of replication, one or moreselectable markers, an enhancer, a polyadenylation signal, and the like.Expression vectors are generally derived from plasmid or viral DNA, ormay contain elements of both.

The term “isolated”, when applied to a polynucleotide, denotes that thepolynucleotide has been removed from its natural genetic milieu and isthus free of other extraneous or unwanted coding sequences, and is in aform suitable for use within genetically engineered protein productionsystems. Such isolated molecules are those that are separated from theirnatural environment and include cDNA and genomic clones. Isolated DNAmolecules of the present invention are free of other genes with whichthey are ordinarily associated, but may include naturally occurring 5′and 3′ untranslated regions such as promoters and terminators. Theidentification of associated regions will be evident to one of ordinaryskill in the art (see for example, Dynan and Tijan, Nature 316:774-78,1985).

An “isolated” polypeptide or protein is a polypeptide or protein that isfound in a condition other than its native environment, such as apartfrom blood and animal tissue. In a preferred form, the isolatedpolypeptide is substantially free of other polypeptides, particularlyother polypeptides of animal origin. It is preferred to provide thepolypeptides in a highly purified form, i.e. greater than 95% pure, morepreferably greater than 99% pure. When used in this context, the term“isolated” does not exclude the presence of the same polypeptide inalternative physical forms, such as dimers or alternatively glycosylatedor derivatized forms.

The term “operably linked”, when referring to DNA segments, denotes thatthe segments are arranged so that they function in concert for theirintended purposes, e.g. transcription initiates in the promoter andproceeds through the coding segment to the terminator.

The term “ortholog” denotes a polypeptide or protein obtained from onespecies that is the functional counterpart of a polypeptide or proteinfrom a different species. Sequence differences among orthologs are theresult of speciation.

“Paralogs” are distinct but structurally related proteins made by anorganism. Paralogs are believed to arise through gene duplication. Forexample, α-globin, β-globin, and myoglobin are paralogs of each other.

The term “polynucleotide” denotes a single- or double-stranded polymerof deoxyribonucleotide or ribonucleotide bases read from the 5′ to the3′ end. Polynucleotides include RNA and DNA, and may be isolated fromnatural sources, synthesized in vitro, or prepared from a combination ofnatural and synthetic molecules. Sizes of polynucleotides are expressedas base pairs (abbreviated “bp”), nucleotides (“nt”), or kilobases(“kb”). Where the context allows, the latter two terms may describepolynucleotides that are single-stranded or double-stranded. When theterm is applied to double-stranded molecules it is used to denoteoverall length and will be understood to be equivalent to the term “basepairs”. It will be recognized by those skilled in the art that the twostrands of a double-stranded polynucleotide may differ slightly inlength and that the ends thereof may be staggered as a result ofenzymatic cleavage; thus all nucleotides within a double-strandedpolynucleotide molecule may not be paired. Such unpaired ends will ingeneral not exceed 20 nt in length.

A “polypeptide” is a polymer of amino acid residues joined by peptidebonds, whether produced naturally or synthetically. Polypeptides of lessthan about 10 amino acid residues are commonly referred to as“peptides”.

“Probes and/or primers” as used herein can be RNA or DNA. DNA can beeither cDNA or genomic DNA. Polynucleotide probes and primers are singleor double-stranded DNA or RNA, generally synthetic oligonucleotides, butmay be generated from cloned cDNA or genomic sequences or itscomplements. Analytical probes will generally be at least 20 nucleotidesin length, although somewhat shorter probes (14-17 nucleotides) can beused. PCR primers are at least 5 nucleotides in length, preferably 15 ormore nt, more preferably 20-30 nt. Short polynucleotides can be usedwhen a small region of the gene is targeted for analysis. For grossanalysis of genes, a polynucleotide probe may comprise an entire exon ormore. Probes can be labeled to provide a detectable signal, such as withan enzyme, biotin, a radionuclide, fluorophore, chemiluminescer,paramagnetic particle and the like, which are commercially availablefrom many sources, such as Molecular Probes, Inc., Eugene, Oreg., andAmersham Corp., Arlington Heights, Ill., using techniques that are wellknown in the art.

The term “promoter” denotes a portion of a gene containing DNA sequencesthat provide for the binding of RNA polymerase and initiation oftranscription. Promoter sequences are commonly, but not always, found inthe 5′ non-coding regions of genes.

The term “receptor” denotes a cell-associated protein that binds to abioactive molecule (i.e., a ligand) and mediates the effect of theligand on the cell. Membrane-bound receptors are characterized by amulti-domain structure comprising an extracellular ligand-binding domainand an intracellular effector domain that is typically involved insignal transduction. Binding of ligand to receptor results in aconformational change in the receptor that causes an interaction betweenthe effector domain and other molecule(s) in the cell. This interactionin turn leads to an alteration in the metabolism of the cell. Metabolicevents that are linked to receptor-ligand interactions include genetranscription, phosphorylation, dephosphorylation, increases in cyclicAMP production, mobilization of cellular calcium, mobilization ofmembrane lipids, cell adhesion, hydrolysis of inositol lipids andhydrolysis of phospholipids. Most nuclear receptors also exhibit amulti-domain structure, including an amino-terminal, transactivatingdomain, a DNA binding domain and a ligand binding domain. In general,receptors can be membrane bound, cytosolic or nuclear; monomeric (e.g.,thyroid stimulating hormone receptor, beta-adrenergic receptor) ormultimeric (e.g., PDGF receptor, growth hormone receptor, IL-3 receptor,GM-CSF receptor, G-CSF receptor, erythropoietin receptor and IL-6receptor).

The term “secretory signal sequence” denotes a DNA sequence that encodesa polypeptide (a “secretory peptide”) that, as a component of a largerpolypeptide, directs the larger polypeptide through a secretory pathwayof a cell in which it is synthesized. The larger peptide is commonlycleaved to remove the secretory peptide during transit through thesecretory pathway.

A “soluble receptor” is a receptor polypeptide that is not bound to acell membrane. Soluble receptors are most commonly ligand-bindingreceptor polypeptides that lack transmembrane and cytoplasmic domains.Soluble receptors can comprise additional amino acid residues, such asaffinity tags that provide for purification of the polypeptide orprovide sites for attachment of the polypeptide to a substrate, orimmunoglobulin constant region sequences. Many cell-surface receptorshave naturally occurring, soluble counterparts that are produced byproteolysis or translated from alternatively spliced mRNAs. Receptorpolypeptides are said to be substantially free of transmembrane andintracellular polypeptide segments when they lack sufficient portions ofthese segments to provide membrane anchoring or signal transduction,respectively.

The term “splice variant” is used herein to denote alternative forms ofRNA transcribed from a gene. Splice variation arises naturally throughuse of alternative splicing sites within a transcribed RNA molecule, orless commonly between separately transcribed RNA molecules, and mayresult in several mRNAs transcribed from the same gene. Splice variantsmay encode polypeptides having altered amino acid sequence. The termsplice variant is also used herein to denote a protein encoded by asplice variant of an mRNA transcribed from a gene.

Molecular weights and lengths of polymers determined by impreciseanalytical methods (e.g., gel electrophoresis) will be understood to beapproximate values. When such a value is expressed as “about” X or“approximately” X, the stated value of X will be understood to beaccurate to ±10%.

All references cited herein are incorporated by reference in theirentirety.

The present invention is based in part upon the discovery of a novel DNAsequence that encodes a polypeptide having homology to an adipocytecomplement related protein (Acrp30). The novel DNA sequence encodes apolypeptide having an amino-terminal signal sequence, an adjacentN-terminal region of non-homology, a collagen domain composed of 34Gly-Xaa-Xaa or Gly-Xaa-Pro repeats and a carboxy-terminal globular-likeC1q domain. The general polypeptide structure set forth above is sharedby Acrp30 and C1q C, except that the collagen-like domain of zacrp2 islonger than that of the other polypeptides. Moreover, the sequencesaligned in the FIGURE share a conserved cysteine residue at position 189of the zacrp2 polypeptide (SEQ ID NO:2). Other regions of homology,found in the carboxy-terminal globular C1q domain in the alignedproteins, are identified herein as useful primers for searching forother family members. Acrp30 and C1q C, for example, would be identifiedin a search using the primers. Also, the zacrp2 polypeptides of thepresent invention include a putative N-linked glycosylation site atamino acid 181 (Asn) of SEQ ID NO:2 and intra-chain disulfide bondingmay involve the cysteine at residue 36 of SEQ ID NO:2.

Analysis of the tissue distribution of the mRNA corresponding to thisnovel DNA using a full length probe showed that zacrp2 is stronglyrepresented in heart, small intestine and colon. Zacrp2 is alsoexpressed in prostate, testis, liver, stomach, thyroid, spinal cord,uterus and trachea. The polypeptide has been designated zacrp2polypeptide.

The novel zacrp2 polypeptides of the present invention were initiallyidentified by querying an EST database for homologs of ACRP30 sequences,characterized by a signal sequence, a collagen-like domain and a C1qdomain. Polypeptides corresponding to ESTs meeting those search criteriawere compared to known sequences to identify proteins having homology toACRP30. An assembled EST cluster was discovered and predicted to be asecreted protein. To identify the corresponding cDNA, a clone consideredlikely to contain the entire coding sequence was used for sequencing.The resulting 1161 bp sequence is disclosed in SEQ ID NO:1. Comparisonof the originally derived EST sequences with the sequence represented inSEQ ID NO:1 showed that there were two frame shifts and a 203 bpinsertion through the collagen-like domain. The novel polypeptideencoded by the full length cDNA enabled the identification of a homologrelationship with adipocyte complement related protein Acrp30 (SEQ IDNO:3) and complement component C1q C (SEQ ID NO:4) as is shown in theFIGURE. Zacrp2 shares 35.5 and 36.5% identity at the amino acid levelwith human C1q C and ACRP30 respectfully. C1q C and ACRP30 share 32%identity. Within the C1q domain, zacrp2 shares 33.6 and 40% identity atthe amino acid level when compared to human C1q C and ACRP30respectfully. C1q C and ACRP30 share 38.2% identity over this region.

The full sequence of the zacrp2 polypeptide was obtained from a singleclone believed to contain it, wherein the clone was obtained from apancreatic tumor tissue library. Other libraries that might also besearched for such clones include heart, small intestine, colon,prostate, testis, liver, stomach, thyroid, spinal cord, uterus trachea,adipose tissue and the like.

The nucleotide sequence of zacrp2 is described in SEQ ID NO:1, and itsdeduced amino acid sequence is described in SEQ ID NO:2. As describedgenerally above, the zacrp2 polypeptide includes a signal sequence,ranging from amino acid 1 (Met) to amino acid residue 15 (Ala). Themature polypeptide therefore ranges from amino acid 16 (Asp) to aminoacid 285 (Val). Within the mature polypeptide, an N-terminal region ofno known homology is found, ranging between amino acid residue 16 (Asp)and 39 (Pro). In addition, a collagen-like domain is found between aminoacid 40 (Gly) and 141 (Cys). In the collagen-like domain, 10 perfectGly-Xaa-Pro and 24 imperfect Gly-Xaa-Xaa repeats are observed. Incontrast, Acrp30 contains 22 perfect or imperfect repeats. Prolineresidues found in this domain at amino acid residue 45, 48, 51, 63, 84,93, 117, 123, 135 and 138 of SEQ ID NO:2 may be hydroxylated. The zacrp2polypeptide also includes a carboxy-terminal C1q domain, ranging fromabout amino acid 142 (Ser) to 285 (Val). Residue 181 (Asn) of SEQ IDNO:2 may be glycosylated. There is a fair amount of conserved structurewithin the C1q domain to enable proper folding. An aromatic motif(F-X(5)-[ND]-X(4)-[FYWL]-X(6)-F-X(5)-G-X-Y-X-F-X-[FY] (SEQ ID NO:5) isalso found within this domain between residues 169 and 199 of SEQ IDNO:2. X represents any amino acid residue and the number in parentheses( ) indicates the amino acid number of residues. The amino acid residuescontained within the square parentheses [] restrict the choice of aminoacid residues at that particular position. Zacrp2 polypeptide, human C1qC and Acrp30 appear to be homologous within the collagen domain and inthe C1q domain, but not in the N-terminal portion of the maturepolypeptide.

Another aspect of the present invention includes zacrp2 polypeptidefragments. Preferred fragments include those containing thecollagen-like domain of zacrp2 polypeptides, ranging from amino acid 1(Met), 16 (Asp) or 40 (Gly) to amino acid 141 (Cys) of SEQ ID NO:2, aportion of the zacrp2 polypeptide containing the collagen-like domain ora portion of the collagen-like domain capable of dimerization oroligomerization. As used herein the term “collagen” or “collagen-likedomain” refers to a series of repeating triplet amino acid sequences,“repeats” or “collagen repeats” represented by the motifs Gly-Xaa-Pro orGly-Xaa-Xaa, where Xaa is any amino acid reside. Such domains maycontain as many as 34 collagen repeats or more. Fragments or proteinscontaining such collagen-like domains may form homomeric constructs(dimers or oligomers of the same fragment or protein). Moreover, suchfragments or proteins containing such collagen-like domains may formheteromeric constructs (dimers, trimers or oligomers of differentfragments or proteins).

These fragments are particularly useful in the study of collagendimerization or oligomerization or in formation of fusion proteins asdescribed more fully below. Polynucleotides encoding such fragments arealso encompassed by the present invention, including the groupconsisting of (a) polynucleotide molecule comprising a sequence ofnucleotides as shown in SEQ ID NO:1 from nucleotide 1, 133, 178 or 250to nucleotide 555; (b) polynucleotide molecules that encode a zacrp2polypeptide fragment that is at least 75% identical to the amino acidsequence of SEQ ID NO:2 from amino acid residue 40 (Gly) to amino acidresidue 141 (Cys); (c) molecules complementary to (a) or (b); and (d)degenerate nucleotide sequences encoding a zacrp2 polypeptidecollagen-like domain fragment.

Other preferred fragments include the globular C1q domain of zacrp2polypeptides, ranging from amino acid 142 (Ser) to 285 (Val) of SEQ IDNO:2, a portion of the zacrp2 polypeptide containing the C1q domain oran active portion of the C1q domain. Other C1q domain containingproteins include C1q A, B and C (Sellar et al., ibid., Reid, ibid., andReid et al., Biochem. J. 203: 559-69, 1982), chipmunkhibernation-associated plasma proteins HP-20, HP-25 and HP-27 (Takamatsuet al., Mol. Cell. Biol. 13: 1516-21, 1993 and Kondo & Kondo, J. Biol.Chem. 267: 473-8, 1992), human precerebellin (Urade et al., Proc. Natl.Acad. Sci. USA 88:1069-73, 1991), human endothelial cell multimerin(Hayward et al., J. Biol. Chem. 270:18246-51, 1995) and vertebratecollagens type VIII and X (Muragaki et al., Eur. J. Biochem. 197:615-22,1991). The globular C1q domain of ACRP30 has been determined to have a10 beta strand “jelly roll” topology (Shapiro and Scherer, Curr. Biol.8:335-8, 1998) that shows significant homology to the TNF family and thezacrp2 sequence as represented by SEQ ID NO:2 contains all 10beta-strands of this structure (amino acid residues 151-155, 172-174,180-183, 187-190, 193-205, 208-214, 220-227, 229-241, 246-251 and269-274 of SEQ ID NO:2). These strands have been designated “A”, “A′”,“B”, “B′”, “C”, “D”, “E”, “F”, “G” and “H” respectively.

Zacrp2 has two receptor binding loops, at amino acid residues 156-182and 214-227 of SEQ ID NO:2. Those skilled in the art will recognize thatthese boundaries are approximate, and are based on alignments with knownproteins and predictions of protein folding. Amino acid residues 193(Gly), 195 (Tyr), 241 (Leu) and 270 (Phe) appear to be conserved acrossthe superfamily including CD40, TNFα, ACRP30 and zacrp2.

These fragments are particularly useful in the study or modulation ofenergy balance or neurotransmission, particularly diet- orstress-related neurotransmission. Anti-microbial activity may also bepresent in such fragments. The homology to TNF proteins suggests suchfragments would be useful in obesity-related insulin resistance, immuneregulation, inflammatory response, apoptosis and osteoclast maturation.Polynucleotides encoding such fragments are also encompassed by thepresent invention, including the group consisting of (a) polynucleotidemolecules comprising a sequence of nucleotides as shown in SEQ ID NO:1from nucleotide 556 to nucleotide 987; (b) polynucleotide molecules thatencode a zacrp2 polypeptide fragment that is at least 80% identical tothe amino acid sequence of SEQ ID NO:2 from amino acid residue 142 (Ser)to amino acid residue 285 (Val); (c) molecules complementary to (a) or(b); and (d) degenerate nucleotide sequences encoding a zacrp2polypeptide C1q domain fragment.

Other zacrp2 polypeptide fragments of the present invention include boththe collagen-like domain and the C1q domain ranging from amino acidresidue 40 (Gly) to 285 (Val) of SEQ ID NO:2. Polynucleotides encodingsuch fragments are also encompassed by the present invention, includingthe group consisting of (a) polynucleotide molecules comprising asequence of nucleotides as shown in SEQ ID NO:1 from nucleotide 250 tonucleotide 987; (b) polynucleotide molecules that encode a zacrp2polypeptide fragment that is at least 80% identical to the amino acidsequence of SEQ ID NO:2 from amino acid residue 40 (Gly) to amino acidresidue 285 (Val); (c) molecules complementary to (a) or (b); and (d)degenerate nucleotide sequences encoding a zacrp2 polypeptidecollagen-like domain-C1q domain fragment.

The highly conserved amino acids, particularly those in thecarboxy-terminal C1q domain of the zacrp2 polypeptide, can be used as atool to identify new family members. For instance, reversetranscription-polymerase chain reaction (RT-PCR) can be used to amplifysequences encoding the conserved motifs from RNA obtained from a varietyof tissue sources. In particular, highly degenerate primers designedfrom conserved sequences are useful for this purpose. In particular, thefollowing primers and their complements are useful for this purpose:

-   Amino acid residues 244-260 of SEQ ID NO:2 GGN GAN SAR GTN TGG YT    (SEQ ID NO:6)-   Amino acid residues 192-197 of SEQ ID NO:2 SN GNN NTN TAY TWY TTY R    (SEQ ID NO:7)-   Amino acid residues 270-275 of SEQ ID NO:2 TTY DSN GGN TTY YTN HT    (SEQ ID NO:8)-   Amino acid residues 179-185 of SEQ ID NO:2 Y TWY RAY RBN WBN WSN GG    (SEQ ID NO:9)    Probes corresponding to complements of the polynucleotides set forth    above are also encompassed.

The present invention also provides polynucleotide molecules, includingDNA and RNA molecules, that encode the zacrp2 polypeptides disclosedherein. Those skilled in the art will readily recognize that, in view ofthe degeneracy of the genetic code, considerable sequence variation ispossible among these polynucleotide molecules. SEQ ID NO:10 is adegenerate DNA sequence that encompasses all DNAs that encode the zacrp2polypeptide of SEQ ID NO:2. Those skilled in the art will recognize thatthe degenerate sequence of SEQ ID NO:10 also provides all RNA sequencesencoding SEQ ID NO:2 by substituting U for T. Thus, zacrp2polypeptide-encoding polynucleotides comprising nucleotide 1 tonucleotide 855 of SEQ ID NO:10 and their RNA equivalents arecontemplated by the present invention. Table 1 sets forth the one-lettercodes used within SEQ ID NO:10 to denote degenerate nucleotidepositions. “Resolutions” are the nucleotides denoted by a code letter.“Complement” indicates the code for the complementary nucleotide(s). Forexample, the code Y denotes either C or T, and its complement R denotesA or G, A being complementary to T, and G being complementary to C.

TABLE 1 Nucleotide Resolution Complement Resolution A A T T C C G G G GC C T T A A R A | G Y C | T Y C | T R A | G M A | C K G | T K G | T M A| C S C | G S C | G W A | T W A | T H A | C | T D A | G | T B C | G | TV A | C | G V A | C | G B C | G | T D A | G | T H A | C | T N A | C | G| T N A | C | G | T

The degenerate codons used in SEQ ID NO:10, encompassing all possiblecodons for a given amino acid, are set forth in Table 2.

TABLE 2 One Amino Letter Degenerate Acid Code Codons Codon Cys C TGC TGTTGY Ser S AGC AGT TCA TCC TCG TCT WSN Thr T ACA ACC ACG ACT ACN Pro PCCA CCC CCG CCT CCN Ala A GCA GCC GCG GCT GCN Gly G GGA GGC GGG GGT GGNAsn N AAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAG GAR Gln Q CAA CAG CARHis H CAC CAT CAY Arg R AGA AGG CGA CGC CGG CGT MGN Lys K AAA AAG AARMet M ATG ATG Ile I ATA ATC ATT ATH Leu L CTA CTC CTG CTT TTA TTG YTNVal V GTA GTC GTG GTT GTN Phe F TTC TTT TTY Tyr Y TAC TAT TAY Trp W TGGTGG Ter . TAA TAG TGA TRR Asn|Asp B RAY Glu|Gln Z SAR Any X NNN

One of ordinary skill in the art will appreciate that some ambiguity isintroduced in determining a degenerate codon, representative of allpossible codons encoding each amino acid. For example, the degeneratecodon for serine (WSN) can, in some circumstances, encode arginine(AGR), and the degenerate codon for arginine (MGN) can, in somecircumstances, encode serine (AGY). A similar relationship existsbetween codons encoding phenylalanine and leucine. Thus, somepolynucleotides encompassed by the degenerate sequence may encodevariant amino acid sequences, but one of ordinary skill in the art caneasily identify such variant sequences by reference to the amino acidsequence of SEQ ID NO:2. Variant sequences can be readily tested forfunctionality as described herein.

One of ordinary skill in the art will also appreciate that differentspecies can exhibit “preferential codon usage.” In general, see,Grantham, et al., Nuc. Acids Res. 8:1893-912, 1980; Haas, et al. Curr.Biol. 6:315-24, 1996; Wain-Hobson, et al., Gene 13:355-64, 1981;Grosjean and Fiers, Gene 18:199-209, 1982; Holm, Nuc. Acids Res.14:3075-87, 1986; Ikemura, J. Mol. Biol. 158:573-97, 1982. As usedherein, the term “preferential codon usage” or “preferential codons” isa term of art referring to protein translation codons that are mostfrequently used in cells of a certain species, thus favoring one or afew representatives of the possible codons encoding each amino acid (SeeTable 2). For example, the amino acid threonine (Thr) may be encoded byACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commonlyused codon; in other species, for example, insect cells, yeast, virusesor bacteria, different Thr codons may be preferential. Preferentialcodons for a particular species can be introduced into thepolynucleotides of the present invention by a variety of methods knownin the art. Introduction of preferential codon sequences intorecombinant DNA can, for example, enhance production of the protein bymaking protein translation more efficient within a particular cell typeor species. Therefore, the degenerate codon sequence disclosed in SEQ IDNO:10 serves as a template for optimizing expression of polynucleotidesin various cell types and species commonly used in the art and disclosedherein. Sequences containing preferential codons can be tested andoptimized for expression in various species, and tested forfunctionality as disclosed herein.

The present invention further provides variant polypeptides and nucleicacid molecules that represent counterparts from other species(orthologs). These species include, but are not limited to mammalian,avian, amphibian, reptile, fish, insect and other vertebrate andinvertebrate species. Of particular interest are zacrp2 polypeptidesfrom other mammalian species, including murine, porcine, ovine, bovine,canine, feline, equine, and other primate polypeptides. A partial murinezacrp2 homolog (SEQ ID NO:12) has been identified. The polynucleotidesequence encoding this murine zacrp2 polypeptide disclosed in SEQ IDNO:11. Orthologs of human zacrp2 can be cloned using information andcompositions provided by the present invention in combination withconventional cloning techniques. For example, a cDNA can be cloned usingmRNA obtained from a tissue or cell type that expresses zacrp2 asdisclosed herein. Suitable sources of mRNA can be identified by probingnorthern blots with probes designed from the sequences disclosed herein.A library is then prepared from mRNA of a positive tissue or cell line.

An zacrp2-encoding cDNA can then be isolated by a variety of methods,such as by probing with a complete or partial human cDNA or with one ormore sets of degenerate probes based on the disclosed sequences. A cDNAcan also be cloned using the polymerase chain reaction with primersdesigned from the representative human zacrp2 sequences disclosedherein. Within an additional method, the cDNA library can be used totransform or transfect host cells, and expression of the cDNA ofinterest can be detected with an antibody to zacrp2 polypeptide. Similartechniques can also be applied to the isolation of genomic clones.

Those skilled in the art will recognize that the sequence disclosed inSEQ ID NO:1 represents a single allele of human zacrp2, and that allelicvariation and alternative splicing are expected to occur. Allelicvariants of this sequence can be cloned by probing cDNA or genomiclibraries from different individuals according to standard procedures.Allelic variants of the nucleotide sequence shown in SEQ ID NO:1,including those containing silent mutations and those in which mutationsresult in amino acid sequence changes, are within the scope of thepresent invention, as are proteins which are allelic variants of SEQ IDNO:2. cDNA molecules generated from alternatively spliced mRNAs, whichretain the properties of the zacrp2 polypeptide are included within thescope of the present invention, as are polypeptides encoded by suchcDNAs and mRNAs. Allelic variants and splice variants of these sequencescan be cloned by probing cDNA or genomic libraries from differentindividuals or tissues according to standard procedures known in theart.

Within preferred embodiments of the invention, the isolated nucleic acidmolecules can hybridize under stringent conditions to nucleic acidmolecules having the nucleotide sequence of SEQ ID NO:1 or to nucleicacid molecules having a nucleotide sequence complementary to SEQ IDNO:1. In general, stringent conditions are selected to be about 5° C.lower than the thermal melting point (T_(m)) for the specific sequenceat a defined ionic strength and pH. The T_(m) is the temperature (underdefined ionic strength and pH) at which 50% of the target sequencehybridizes to a perfectly matched probe.

A pair of nucleic acid molecules, such as DNA-DNA, RNA-RNA and DNA-RNA,can hybridize if the nucleotide sequences have some degree ofcomplementarity. Hybrids can tolerate mismatched base pairs in thedouble helix, but the stability of the hybrid is influenced by thedegree of mismatch. The T_(m) of the mismatched hybrid decreases by 1°C. for every 1-1.5% base pair mismatch. Varying the stringency of thehybridization conditions allows control over the degree of mismatch thatwill be present in the hybrid. The degree of stringency increases as thehybridization temperature increases and the ionic strength of thehybridization buffer decreases. Stringent hybridization conditionsencompass temperatures of about 5-25° C. below the T_(m) of the hybridand a hybridization buffer having up to 1 M Na⁺. Higher degrees ofstringency at lower temperatures can be achieved with the addition offormamide which reduces the T_(m) of the hybrid about 1° C. for each 1%formamide in the buffer solution. Generally, such stringent conditionsinclude temperatures of 20-70° C. and a hybridization buffer containingup to 6×SSC and 0-50% formamide. A higher degree of stringency can beachieved at temperatures of from 40-70° C. with a hybridization bufferhaving up to 4×SSC and from 0-50% formamide. Highly stringent conditionstypically encompass temperatures of 42-70° C. with a hybridizationbuffer having up to 1×SSC and 0-50% formamide. Different degrees ofstringency can be used during hybridization and washing to achievemaximum specific binding to the target sequence. Typically, the washesfollowing hybridization are performed at increasing degrees ofstringency to remove non-hybridized polynucleotide probes fromhybridized complexes.

The above conditions are meant to serve as a guide and it is well withinthe abilities of one skilled in the art to adapt these conditions foruse with a particular polypeptide hybrid. The T_(m) for a specifictarget sequence is the temperature (under defined conditions) at which50% of the target sequence will hybridize to a perfectly matched probesequence. Those conditions which influence the T_(m) include, the sizeand base pair content of the polynucleotide probe, the ionic strength ofthe hybridization solution, and the presence of destabilizing agents inthe hybridization solution. Numerous equations for calculating T_(m) areknown in the art, and are specific for DNA, RNA and DNA-RNA hybrids andpolynucleotide probe sequences of varying length (see, for example,Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition(Cold Spring Harbor Press 1989); Ausubel et al., (eds.), CurrentProtocols in Molecular Biology (John Wiley and Sons, Inc. 1987); Bergerand Kimmel (eds.), Guide to Molecular Cloning Techniques, (AcademicPress, Inc. 1987); and Wetmur, Crit. Rev. Biochem. Mol. Biol. 26:227(1990)). Sequence analysis software, such as OLIGO 6.0 (LSR; Long Lake,Minn.) and Primer Premier 4.0 (Premier Biosoft International; Palo Alto,Calif.), as well as sites on the Internet, are available tools foranalyzing a given sequence and calculating T_(m) based on user definedcriteria. Such programs can also analyze a given sequence under definedconditions and identify suitable probe sequences. Typically,hybridization of longer polynucleotide sequences, >50 base pairs, isperformed at temperatures of about 20-25° C. below the calculated T_(m).For smaller probes, <50 base pairs, hybridization is typically carriedout at the T_(m) or 5-10° C. below. This allows for the maximum rate ofhybridization for DNA-DNA and DNA-RNA hybrids.

The length of the polynucleotide sequence influences the rate andstability of hybrid formation. Smaller probe sequences, <50 base pairs,reach equilibrium with complementary sequences rapidly, but may formless stable hybrids. Incubation times of anywhere from minutes to hourscan be used to achieve hybrid formation. Longer probe sequences come toequilibrium more slowly, but form more stable complexes even at lowertemperatures. Incubations are allowed to proceed overnight or longer.Generally, incubations are carried out for a period equal to three timesthe calculated Cot time. Cot time, the time it takes for thepolynucleotide sequences to reassociate, can be calculated for aparticular sequence by methods known in the art.

The base pair composition of polynucleotide sequence will effect thethermal stability of the hybrid complex, thereby influencing the choiceof hybridization temperature and the ionic strength of the hybridizationbuffer. A-T pairs are less stable than G-C pairs in aqueous solutionscontaining sodium chloride. Therefore, the higher the G-C content, themore stable the hybrid. Even distribution of G and C residues within thesequence also contribute positively to hybrid stability. In addition,the base pair composition can be manipulated to alter the T_(m) of agiven sequence. For example, 5-methyldeoxycytidine can be substitutedfor deoxycytidine and 5-bromodeoxuridine can be substituted forthymidine to increase the T_(m), whereas 7-deazz-2′-deoxyguanosine canbe substituted for guanosine to reduce dependence on T_(m).

The ionic concentration of the hybridization buffer also affects thestability of the hybrid. Hybridization buffers generally containblocking agents such as Denhardt's solution (Sigma Chemical Co., St.Louis, Mo.), denatured salmon sperm DNA, tRNA, milk powders (BLOTTO),heparin or SDS, and a Na⁺ source, such as SSC (1×SSC: 0.15 M sodiumchloride, 15 mM sodium citrate) or SSPE (1×SSPE: 1.8 M NaCl, 10 mMNaH₂PO₄, 1 mM EDTA, pH 7.7). By decreasing the ionic concentration ofthe buffer, the stability of the hybrid is increased. Typically,hybridization buffers contain from between 10 mM-1 M Na⁺. The additionof destabilizing or denaturing agents such as formamide,tetralkylammonium salts, guanidinium cations or thiocyanate cations tothe hybridization solution will alter the T_(m) of a hybrid. Typically,formamide is used at a concentration of up to 50% to allow incubationsto be carried out at more convenient and lower temperatures. Formamidealso acts to reduce non-specific background when using RNA probes.

As an illustration, a nucleic acid molecule encoding a variant zacrp2polypeptide can be hybridized with a nucleic acid molecule having thenucleotide sequence of SEQ ID NO:1 (or its complement) at 42° C.overnight in a solution comprising 50% formamide, 5×SSC (1×SSC: 0.15 Msodium chloride and 15 mM sodium citrate), 50 mM sodium phosphate (pH7.6), 5× Denhardt's solution (100× Denhardt's solution: 2% (w/v) Ficoll400, 2% (w/v) polyvinylpyrrolidone, and 2% (w/v) bovine serum albumin),10% dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA.One of skill in the art can devise variations of these hybridizationconditions. For example, the hybridization mixture can be incubated at ahigher or lower temperature, such as about 65° C., in a solution thatdoes not contain formamide. Moreover, premixed hybridization solutionsare available (e.g., EXPRESSHYB Hybridization Solution from CLONTECHLaboratories, Inc.), and hybridization can be performed according to themanufacturer's instructions.

Following hybridization, the nucleic acid molecules can be washed toremove non-hybridized nucleic acid molecules under stringent conditions,or under highly stringent conditions. Typical stringent washingconditions include washing in a solution of 0.5×-2×SSC with 0.1% sodiumdodecyl sulfate (SDS) at 50-65° C. That is, nucleic acid moleculesencoding a variant zacrp2 polypeptide hybridize with a nucleic acidmolecule having the nucleotide sequence of SEQ ID NO:1 (or itscomplement) under stringent washing conditions, in which the washstringency is equivalent to 0.5×-2×SSC with 0.1% SDS at 50-65° C.,including 0.5×SSC with 0.1% SDS at 55° C., or 2×SSC with 0.1% SDS at 65°C. One of skill in the art can readily devise equivalent conditions, forexample, by substituting SSPE for SSC in the wash solution.

Typical highly stringent washing conditions include washing in asolution of 0.1×-0.2×SSC with 0.1% sodium dodecyl sulfate (SDS) at50-65° C. In other words, nucleic acid molecules encoding a variantzacrp2 polypeptide hybridize with a nucleic acid molecule having thenucleotide sequence of SEQ ID NO:1 (or its complement) under highlystringent washing conditions, in which the wash stringency is equivalentto 0.1×-0.2×SSC with 0.1% SDS at 50-65° C., including 0.1×SSC with 0.1%SDS at 50° C., or 0.2×SSC with 0.1% SDS at 65° C.

The present invention also provides isolated zacrp2 polypeptides thathave a substantially similar sequence identity to the polypeptides ofSEQ ID NO:2, or their orthologs. The term “substantially similarsequence identity” is used herein to denote polypeptides having at least70%, at least 80%, at least 90%, at least 95% or greater than 95%sequence identity to the sequences shown in SEQ ID NO:2, or theirorthologs. The present invention also includes polypeptides thatcomprise an amino acid sequence having at least 70%, at least 80%, atleast 90%, at least 95% or greater than 95% sequence identity to thesequence of amino acid residues 40 to 285 of SEQ ID NO:2. The presentinvention further includes nucleic acid molecules that encode suchpolypeptides. Methods for determining percent identity are describedbelow.

The present invention also contemplates zacrp2 variant nucleic acidmolecules that can be identified using two criteria: a determination ofthe similarity between the encoded polypeptide with the amino acidsequence of SEQ ID NO:2, and a hybridization assay, as described above.Such zacrp2 variants include nucleic acid molecules (1) that hybridizewith a nucleic acid molecule having the nucleotide sequence of SEQ IDNO:1 (or its complement) under stringent washing conditions, in whichthe wash stringency is equivalent to 0.5×-2×SSC with 0.1% SDS at 55-65°C., and (2) that encode a polypeptide having at least 70%, at least 80%,at least 90%, at least 95% or greater than 95% sequence identity to theamino acid sequence of SEQ ID NO:2. Alternatively, zacrp2 variants canbe characterized as nucleic acid molecules (1) that hybridize with anucleic acid molecule having the nucleotide sequence of SEQ ID NO:1 (orits complement) under highly stringent washing conditions, in which thewash stringency is equivalent to 0.1×-0.2×SSC with 0.1% SDS at 50-65°C., and (2) that encode a polypeptide having at least 70%, at least 80%,at least 90%, at least 95% or greater than 95% sequence identity to theamino acid sequence of SEQ ID NO:2.

Percent sequence identity is determined by conventional methods. See,for example, Altschul et al., Bull. Math. Bio. 48:603, 1986, andHenikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1992.Briefly, two amino acid sequences are aligned to optimize the alignmentscores using a gap opening penalty of 10, a gap extension penalty of 1,and the “BLOSUM62” scoring matrix of Henikoff and Henikoff (ibid.) asshown in Table 3 (amino acids are indicated by the standard one-lettercodes). The percent identity is then calculated as: ([Total number ofidentical matches]/[length of the longer sequence plus the number ofgaps introduced into the longer sequence in order to align the twosequences])(100).

TABLE 3 A R N D C Q E G H I L K M F P S T W Y V A 4 R −1 5 N −2 0 6 D −2−2 1 6 C 0 −3 −3 −3 9 Q −1 1 0 0 −3 5 E −1 0 0 2 −4 2 5 G 0 −2 0 −1 −3−2 −2 6 H −2 0 1 −1 −3 0 0 −2 8 I −1 −3 −3 −3 −1 −3 −3 −4 −3 4 L −1 −2−3 −4 −l −2 −3 −4 −3 2 4 K −1 2 0 −1 −3 1 1 −2 −1 −3 −2 5 M −1 −1 −2 −3−1 0 −2 −3 −2 1 2 −1 5 F −2 −3 −3 −3 −2 −3 −3 −3 −1 0 0 −3 0 6 P −1 −2−2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 7 S 1 −1 1 0 −1 0 0 0 −1 −2 −2 0 −1−2 −1 4 T 0 −1 0 −1 −1 −1 −1 −2 −2 −1 −1 −1 −1 −2 −1 1 5 W −3 −3 −4 −4−2 −2 −3 −2 −2 −3 −2 −3 −1 1 −4 −3 −2 11 Y −2 −2 −2 −3 −2 −1 −2 −3 2 −1−1 −2 −1 3 −3 −2 −2 2 7 V 0 −3 −3 −3 −1 −2 −2 −3 −3 3 1 −2 1 −1 −2 −2 0−3 −1 4

Those skilled in the art appreciate that there are many establishedalgorithms available to align two amino acid sequences. The “FASTA”similarity search algorithm of Pearson and Lipman is a suitable proteinalignment method for examining the level of identity shared by an aminoacid sequence disclosed herein and the amino acid sequence of a putativevariant zacrp2. The FASTA algorithm is described by Pearson and Lipman,Proc. Nat. Acad. Sci. USA 85:2444, 1988, and by Pearson, Meth. Enzymol.183:63, 1990.

Briefly, FASTA first characterizes sequence similarity by identifyingregions shared by the query sequence (e.g., SEQ ID NO:2) and a testsequence that have either the highest density of identities (if the ktupvariable is 1) or pairs of identities (if ktup=2), without consideringconservative amino acid substitutions, insertions, or deletions. The tenregions with the highest density of identities are then re-scored bycomparing the similarity of all paired amino acids using an amino acidsubstitution matrix, and the ends of the regions are “trimmed” toinclude only those residues that contribute to the highest score. Ifthere are several regions with scores greater than the “cutoff” value(calculated by a predetermined formula based upon the length of thesequence and the ktup value), then the trimmed initial regions areexamined to determine whether the regions can be joined to form anapproximate alignment with gaps. Finally, the highest scoring regions ofthe two amino acid sequences are aligned using a modification of theNeedleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol. Biol.48:444, 1970; Sellers, SIAM J. Appl. Math. 26:787, 1974), which allowsfor amino acid insertions and deletions. Illustrative parameters forFASTA analysis are: ktup=1, gap opening penalty=10, gap extensionpenalty=1, and substitution matrix=BLOSUM62. These parameters can beintroduced into a FASTA program by modifying the scoring matrix file(“SMATRIX”), as explained in Appendix 2 of Pearson, Meth. Enzymol.183:63, 1990.

FASTA can also be used to determine the sequence identity of nucleicacid molecules using a ratio as disclosed above. For nucleotide sequencecomparisons, the ktup value can range between one to six, preferablyfrom four to six.

The present invention includes nucleic acid molecules that encode apolypeptide having one or more “conservative amino acid substitutions,”compared with the amino acid sequence of SEQ ID NO:2. Conservative aminoacid substitutions can be based upon the chemical properties of theamino acids. That is, variants can be obtained that contain one or moreamino acid substitutions of SEQ ID NO:2, in which an alkyl amino acid issubstituted for an alkyl amino acid in a zacrp2 amino acid sequence, anaromatic amino acid is substituted for an aromatic amino acid in azacrp2 amino acid sequence, a sulfur-containing amino acid issubstituted for a sulfur-containing amino acid in a zacrp2 amino acidsequence, a hydroxy-containing amino acid is substituted for ahydroxy-containing amino acid in a zacrp2 amino acid sequence, an acidicamino acid is substituted for an acidic amino acid in a zacrp2 aminoacid sequence, a basic amino acid is substituted for a basic amino acidin a zacrp2 amino acid sequence, or a dibasic monocarboxylic amino acidis substituted for a dibasic monocarboxylic amino acid in a zacrp2 aminoacid sequence.

Among the common amino acids, for example, a “conservative amino acidsubstitution” is illustrated by a substitution among amino acids withineach of the following groups: (1) glycine, alanine, valine, leucine, andisoleucine, (2) phenylalanine, tyrosine, and tryptophan, (3) serine andthreonine, (4) aspartate and glutamate, (5) glutamine and asparagine,and (6) lysine, arginine and histidine.

The BLOSUM62 table is an amino acid substitution matrix derived fromabout 2,000 local multiple alignments of protein sequence segments,representing highly conserved regions of more than 500 groups of relatedproteins (Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915,1992). Accordingly, the BLOSUM62 substitution frequencies can be used todefine conservative amino acid substitutions that may be introduced intothe amino acid sequences of the present invention. Although it ispossible to design amino acid substitutions based solely upon chemicalproperties (as discussed above), the language “conservative amino acidsubstitution” preferably refers to a substitution represented by aBLOSUM62 value of greater than −1. For example, an amino acidsubstitution is conservative if the substitution is characterized by aBLOSUM62 value of 0, 1, 2, or 3. According to this system, preferredconservative amino acid substitutions are characterized by a BLOSUM62value of at least 1 (e.g., 1, 2 or 3), while more preferred conservativeamino acid substitutions are characterized by a BLOSUM62 value of atleast 2 (e.g., 2 or 3).

Conservative amino acid changes in a zacrp2 gene can be introduced bysubstituting nucleotides for the nucleotides recited in SEQ ID NO:1.Such “conservative amino acid” variants can be obtained, for example, byoligonucleotide-directed mutagenesis, linker-scanning mutagenesis,mutagenesis using the polymerase chain reaction, and the like (seeAusubel (1995) at pages 8-10 to 8-22; and McPherson (ed.), DirectedMutagenesis: A Practical Approach (IRL Press 1991)). The ability of suchvariants to promote the energy balance modulating or other properties ofthe wild-type protein can be determined using a standard methods, suchas the assays described herein. Alternatively, a variant zacrp2polypeptide can be identified by the ability to specifically bindanti-zacrp2 antibodies.

The proteins of the present invention can also comprise non-naturallyoccurring amino acid residues. Non-naturally occurring amino acidsinclude, without limitation, trans-3-methylproline, 2,4-methanoproline,cis-4-hydroxyproline, trans-4-hydroxyproline, N-methyl-glycine,allo-threonine, methylthreonine, hydroxyethyl-cysteine,hydroxyethylhomocysteine, nitroglutamine, homo-glutamine, pipecolicacid, thiazolidine carboxylic acid, dehydroproline, 3- and4-methylproline, 3,3-dimethyl-proline, tert-leucine, norvaline,2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and4-fluorophenyl-alanine. Several methods are known in the art forincorporating non-naturally occurring amino acid residues into proteins.For example, an in vitro system can be employed wherein nonsensemutations are suppressed using chemically aminoacylated suppressortRNAs. Methods for synthesizing amino acids and aminoacylating tRNA areknown in the art. Transcription and translation of plasmids containingnonsense mutations is typically carried out in a cell-free systemcomprising an E. coli S30 extract and commercially available enzymes andother reagents. Proteins are purified by chromatography. See, forexample, Robertson et al., J. Am. Chem. Soc. 113:2722, 1991, Ellman etal., Methods Enzymol. 202:301, 1991, Chung et al., Science 259:806,1993, and Chung et al., Proc. Nat. Acad. Sci. USA 90:10145, 1993.

In a second method, translation is carried out in Xenopus oocytes bymicroinjection of mutated mRNA and chemically aminoacylated suppressortRNAs (Turcatti et al., J. Biol. Chem. 271:19991, 1996). Within a thirdmethod, E. coli cells are cultured in the absence of a natural aminoacid that is to be replaced (e.g., phenylalanine) and in the presence ofthe desired non-naturally occurring amino acid(s) (e.g.,2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or4-fluorophenylalanine). The non-naturally occurring amino acid isincorporated into the protein in place of its natural counterpart. See,Koide et al., Biochem. 33:7470, 1994. Naturally occurring amino acidresidues can be converted to non-naturally occurring species by in vitrochemical modification. Chemical modification can be combined withsite-directed mutagenesis to further expand the range of substitutions(Wynn and Richards, Protein Sci. 2:395, 1993).

A limited number of non-conservative amino acids, amino acids that arenot encoded by the genetic code, non-naturally occurring amino acids,and unnatural amino acids may be substituted for zacrp2 amino acidresidues.

Multiple amino acid substitutions can be made and tested using knownmethods of mutagenesis and screening, such as those disclosed byReidhaar-Olson and Sauer (Science 241:53, 1988) or Bowie and Sauer(Proc. Nat. Acad. Sci. USA 86:2152, 1989). Briefly, these authorsdisclose methods for simultaneously randomizing two or more positions ina polypeptide, selecting for functional polypeptide, and then sequencingthe mutagenized polypeptides to determine the spectrum of allowablesubstitutions at each position. Other methods that can be used includephage display (e.g., Lowman et al., Biochem. 30:10832, 1991, Ladner etal., U.S. Pat. No. 5,223,409, Huse, international publication No. WO92/06204, and region-directed mutagenesis (Derbyshire et al., Gene46:145, 1986, and Ner et al., DNA 7:127, 1988).

Variants of the disclosed zacrp2 nucleotide and polypeptide sequencescan also be generated through DNA shuffling as disclosed by Stemmer,Nature 370:389, 1994, Stemmer, Proc. Nat. Acad. Sci. USA 91:10747, 1994,and international publication No. WO 97/20078. Briefly, variant DNAmolecules are generated by in vitro homologous recombination by randomfragmentation of a parent DNA followed by reassembly using PCR,resulting in randomly introduced point mutations. This technique can bemodified by using a family of parent DNA molecules, such as allelicvariants or DNA molecules from different species, to introduceadditional variability into the process. Selection or screening for thedesired activity, followed by additional iterations of mutagenesis andassay provides for rapid “evolution” of sequences by selecting fordesirable mutations while simultaneously selecting against detrimentalchanges.

Mutagenesis methods as disclosed herein can be combined withhigh-throughput, automated screening methods to detect activity ofcloned, mutagenized polypeptides in host cells. Mutagenized DNAmolecules that encode biologically active polypeptides, or polypeptidesthat bind with anti-zacrp2 antibodies, can be recovered from the hostcells and rapidly sequenced using modern equipment. These methods allowthe rapid determination of the importance of individual amino acidresidues in a polypeptide of interest, and can be applied topolypeptides of unknown structure.

Essential amino acids in the polypeptides of the present invention canbe identified according to procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (Cunninghamand Wells, Science 244:1081, 1989, Bass et al., Proc. Nat. Acad. Sci.USA 88:4498, 1991, Coombs and Corey, “Site-Directed Mutagenesis andProtein Engineering,” in Proteins: Analysis and Design, Angeletti (ed.),pages 259-311 (Academic Press, Inc. 1998)). In the latter technique,single alanine mutations are introduced at every residue in themolecule, and the resultant mutant molecules are tested for biologicalactivity as disclosed below to identify amino acid residues that arecritical to the activity of the molecule. See also, Hilton et al., J.Biol. Chem. 271:4699, 1996. The identities of essential amino acids canalso be inferred from analysis of homologies with zacrp2.

The location of zacrp2 receptor binding domains can be identified byphysical analysis of structure, as determined by such techniques asnuclear magnetic resonance, crystallography, electron diffraction orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids. See, for example, de Vos et al., Science 255:306,1992, Smith et al., J. Mol. Biol. 224:899, 1992, and Wlodaver et al.,FEBS Lett. 309:59, 1992. Moreover, zacrp2 labeled with biotin or FITCcan be used for expression cloning of zacrp2 receptors.

The present invention also provides polypeptide fragments or peptidescomprising an epitope-bearing portion of a zacrp2 polypeptide describedherein. Such fragments or peptides may comprise an “immunogenicepitope,” which is a part of a protein that elicits an antibody responsewhen the entire protein is used as an immunogen. Immunogenicepitope-bearing peptides can be identified using standard methods (see,for example, Geysen et al., Proc. Nat. Acad. Sci. USA 81:3998, 1983).

In contrast, polypeptide fragments or peptides may comprise an“antigenic epitope,” which is a region of a protein molecule to which anantibody can specifically bind. Certain epitopes consist of a linear orcontiguous stretch of amino acids, and the antigenicity of such anepitope is not disrupted by denaturing agents. It is known in the artthat relatively short synthetic peptides that can mimic epitopes of aprotein can be used to stimulate the production of antibodies againstthe protein (see, for example, Sutcliffe et al., Science 219:660, 1983).Accordingly, antigenic epitope-bearing peptides and polypeptides of thepresent invention are useful to raise antibodies that bind with thepolypeptides described herein.

Antigenic epitope-bearing peptides and polypeptides preferably containat least four to ten amino acids, at least ten to fifteen amino acids,or about 15 to about 30 amino acids of SEQ ID NO:2. Such epitope-bearingpeptides and polypeptides can be produced by fragmenting a zacrp2polypeptide, or by chemical peptide synthesis, as described herein.Moreover, epitopes can be selected by phage display of random peptidelibraries (see, for example, Lane and Stephen, Curr. Opin. Immunol.5:268, 1993, and Cortese et al., Curr. Opin. Biotechnol. 7:616, 1996).Standard methods for identifying epitopes and producing antibodies fromsmall peptides that comprise an epitope are described, for example, byMole, “Epitope Mapping,” in Methods in Molecular Biology, Vol. 10,Manson (ed.), pages 105-16 (The Humana Press, Inc. 1992), Price,“Production and Characterization of Synthetic Peptide-DerivedAntibodies,” in Monoclonal Antibodies: Production, Engineering, andClinical Application, Ritter and Ladyman (eds.), pages 60-84 (CambridgeUniversity Press 1995), and Coligan et al. (eds.), Current Protocols inImmunology, pages 9.3.1-9.3.5 and pages 9.4.1-9.4.11 (John Wiley & Sons1997).

Regardless of the particular nucleotide sequence of a variant zacrp2gene, the gene encodes a polypeptide that is characterized by its energybalance modulating activity or other activities of the wild-typeprotein, or by the ability to bind specifically to an anti-zacrp2antibody. More specifically, variant zacrp2 genes encode polypeptideswhich exhibit at least 50%, and preferably, greater than 70, 80, or 90%,of the activity of polypeptide encoded by the human zacrp2 genedescribed herein.

For any zacrp2 polypeptide, including variants and fusion proteins, oneof ordinary skill in the art can readily generate a fully degeneratepolynucleotide sequence encoding that variant using the information setforth in Tables 1 and 2 above. Moreover, those of skill in the art canuse standard software to devise zacrp2 variants based upon thenucleotide and amino acid sequences described herein. Accordingly, thepresent invention includes a computer-readable medium encoded with adata structure that provides at least one of the following sequences:SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:10. Suitable forms ofcomputer-readable media include magnetic media and optically-readablemedia. Examples of magnetic media include a hard or fixed drive, arandom access memory (RAM) chip, a floppy disk, digital linear tape(DLT), a disk cache, and a ZIP disk. Optically readable media areexemplified by compact discs (e.g., CD-read only memory (ROM),CD-rewritable (RW), and CD-recordable), and digital versatile/videodiscs (DVD) (e.g., DVD-ROM, DVD-RAM, and DVD+RW).

The present invention also provides zacrp2 fusion proteins. For example,fusion proteins of the present invention encompass (1) a polypeptideselected from the group consisting of: (a) polypeptide moleculescomprising a sequence of amino acid residues as shown in SEQ ID NO:2from amino acid residue 1 (Met), 16 (Asp) or 40 (Gly) to amino acidresidue 285 (Val); (b) polypeptide molecules ranging from amino acid 40(Gly) to amino acid 141 (Cys) of SEQ ID NO:2, a portion of the zacrp2polypeptide containing the collagen-like domain or a portion of thecollagen-like domain capable of dimerization or oligomerization; (c)polypeptide molecules ranging from amino acid 142 (Ser) to 285 (Val) ofSEQ ID NO:2, a portion of the zacrp2 polypeptide containing the C1qdomain or an active portion of the C1q domain; or (d) polypeptidemolecules ranging from amino acid 40 (Gly) to 285 (Val), a portion ofthe zacrp2 polypeptide including the collagen-like domain and the C1qdomain; and (2) another polypeptide. The other polypeptide may bealternative or additional C1q domain, an alternative or additionalcollagen-like domain, a signal peptide to facilitate secretion of thefusion protein or the like. The globular domain of complement binds IgG,thus, the globular domain of zacrp2 polypeptide, fragment or fusion mayhave a similar role.

Zacrp2 polypeptides, ranging from amino acid 1 (Met) to amino acid 285(Val); the mature zacrp2 polypeptides, ranging from amino acid 16 (Asp)to amino acid 285 (Val); or the secretion leader fragments thereof,which fragments range from amino acid 1 (Met) to amino acid 15 (Ala) maybe used in the study of secretion of proteins from cells. In preferredembodiments of this aspect of the present invention, the maturepolypeptides are formed as fusion proteins with putative secretorysignal sequences; plasmids bearing regulatory regions capable ofdirecting the expression of the fusion protein is introduced into testcells; and secretion of mature protein is monitored. The monitoring maybe done by techniques known in the art, such as HPLC and the like.

The polypeptides of the present invention, including full-lengthproteins, fragments thereof and fusion proteins, can be produced ingenetically engineered host cells according to conventional techniques.Suitable host cells are those cell types that can be transformed ortransfected with exogenous DNA and grown in culture, and includebacteria, fungal cells, and cultured higher eukaryotic cells. Eukaryoticcells, particularly cultured cells of multicellular organisms, arepreferred. Techniques for manipulating cloned DNA molecules andintroducing exogenous DNA into a variety of host cells are disclosed bySambrook et al., ibid., and Ausubel et al. ibid.

In general, a DNA sequence encoding a zacrp2 polypeptide of the presentinvention is operably linked to other genetic elements required for itsexpression, generally including a transcription promoter and terminatorwithin an expression vector. The vector will also commonly contain oneor more selectable markers and one or more origins of replication,although those skilled in the art will recognize that within certainsystems selectable markers may be provided on separate vectors, andreplication of the exogenous DNA may be provided by integration into thehost cell genome. Selection of promoters, terminators, selectablemarkers, vectors and other elements is a matter of routine design withinthe level of ordinary skill in the art. Many such elements are describedin the literature and are available through commercial suppliers.

To direct a zacrp2 polypeptide into the secretory pathway of a hostcell, a secretory signal sequence (also known as a leader sequence,signal sequence, prepro sequence or pre-sequence) is provided in theexpression vector. The secretory signal sequence may be that of thezacrp2 polypeptide, or may be derived from another secreted protein(e.g., t-PA) or synthesized de novo. The secretory signal sequence isjoined to the zacrp2 polypeptide DNA sequence in the correct readingframe. Secretory signal sequences are commonly positioned 5′ to the DNAsequence encoding the polypeptide of interest, although certain signalsequences may be positioned elsewhere in the DNA sequence of interest(see, e.g., Welch et al., U.S. Pat. No. 5,037,743; Holland et al., U.S.Pat. No. 5,143,830). Conversely, the signal sequence portion of thezacrp2 polypeptide (amino acids 1-15 of SEQ ID NO:2) may be employed todirect the secretion of an alternative protein by analogous methods.

The secretory signal sequence contained in the polypeptides of thepresent invention can be used to direct other polypeptides into thesecretory pathway. The present invention provides for such fusionpolypeptides. A signal fusion polypeptide can be made wherein asecretory signal sequence derived from amino acid residues 1-15 of SEQID NO:2 is operably linked to another polypeptide using methods known inthe art and disclosed herein. The secretory signal sequence contained inthe fusion polypeptides of the present invention is preferably fusedamino-terminally to an additional peptide to direct the additionalpeptide into the secretory pathway. Such constructs have numerousapplications known in the art. For example, these novel secretory signalsequence fusion constructs can direct the secretion of an activecomponent of a normally non-secreted protein, such as a receptor. Suchfusions may be used in vivo or in vitro to direct peptides through thesecretory pathway.

Cultured mammalian cells are suitable hosts within the presentinvention. Methods for introducing exogenous DNA into mammalian hostcells include calcium phosphate-mediated transfection (Wigler et al.,Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603,1981: Graham and Van der Eb, Virology 52:456, 1973), electroporation(Neumann et al., EMBO J. 1:841-5, 1982), DEAE-dextran mediatedtransfection (Ausubel et al., ibid.), and liposome-mediated transfection(Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80,1993, and viral vectors (Miller and Rosman, BioTechniques 7:980-90,1989; Wang and Finer, Nature Med. 2:714-6, 1996). The production ofrecombinant polypeptides in cultured mammalian cells is disclosed, forexample, by Levinson et al., U.S. Pat. No. 4,713,339; Hagen et al., U.S.Pat. No. 4,784,950; Palmiter et al., U.S. Pat. No. 4,579,821; andRingold, U.S. Pat. No. 4,656,134. Suitable cultured mammalian cellsinclude the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK(ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) and Chinese hamsterovary (e.g. CHO-K1; ATCC No. CCL 61) cell lines. Additional suitablecell lines are known in the art and available from public depositoriessuch as the American Type Culture Collection, Manassas, Va. In general,strong transcription promoters are preferred, such as promoters fromSV-40 or cytomegalovirus. See, e.g., U.S. Pat. No. 4,956,288. Othersuitable promoters include those from metallothionein genes (U.S. Pat.Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.

Drug selection is generally used to select for cultured mammalian cellsinto which foreign DNA has been inserted. Such cells are commonlyreferred to as “transfectants”. Cells that have been cultured in thepresence of the selective agent and are able to pass the gene ofinterest to their progeny are referred to as “stable transfectants.” Apreferred selectable marker is a gene encoding resistance to theantibiotic neomycin. Selection is carried out in the presence of aneomycin-type drug, such as G-418 or the like. Selection systems mayalso be used to increase the expression level of the gene of interest, aprocess referred to as “amplification.” Amplification is carried out byculturing transfectants in the presence of a low level of the selectiveagent and then increasing the amount of selective agent to select forcells that produce high levels of the products of the introduced genes.A preferred amplifiable selectable marker is dihydrofolate reductase,which confers resistance to methotrexate. Other drug resistance genes(e.g., hygromycin resistance, multi-drug resistance, puromycinacetyltransferase) can also be used. Alternative markers that introducean altered phenotype, such as green fluorescent protein, or cell surfaceproteins such as CD4, CD8, Class I MHC, placental alkaline phosphatasemay be used to sort transfected cells from untransfected cells by suchmeans as FACS sorting or magnetic bead separation technology.

Other higher eukaryotic cells can also be used as hosts, including plantcells, insect cells and avian cells. The use of Agrobacterium rhizogenesas a vector for expressing genes in plant cells has been reviewed bySinkar et al., J. Biosci. (Bangalore) 11:47-58, 1987. Transformation ofinsect cells and production of foreign polypeptides therein is disclosedby Guarino et al., U.S. Pat. No. 5,162,222 and WIPO publication WO94/06463. Insect cells can be infected with recombinant baculovirus,commonly derived from Autographa californica nuclear polyhedrosis virus(AcNPV). See, King and Possee, The Baculovirus Expression System: ALaboratory Guide, London, Chapman & Hall; O'Reilly et al., BaculovirusExpression Vectors: A Laboratory Manual, New York, Oxford UniversityPress., 1994; and, Richardson, C. D., Ed., Baculovirus ExpressionProtocols. Methods in Molecular Biology, Totowa, N.J., Humana Press,1995. A second method of making recombinant zacrp2 baculovirus utilizesa transposon-based system described by Luckow (Luckow et al., J. Virol.67:4566-79, 1993). This system, which utilizes transfer vectors, is soldin the Bac-to-Bac™ kit (Life Technologies, Rockville, Md.). This systemutilizes a transfer vector, pFastBac1™ (Life Technologies) containing aTn7 transposon to move the DNA encoding the zacrp2 polypeptide into abaculovirus genome maintained in E. coli as a large plasmid called a“bacmid.” The pFastBac1™ transfer vector utilizes the AcNPV polyhedrinpromoter to drive the expression of the gene of interest, in this casezacrp2. However, pFastBac1™ can be modified to a considerable degree.The polyhedrin promoter can be removed and substituted with thebaculovirus basic protein promoter (also known as Pcor, p6.9 or MPpromoter) which is expressed earlier in the baculovirus infection, andhas been shown to be advantageous for expressing secreted proteins. See,Hill-Perkins and Possee, J. Gen. Virol. 71:971-6, 1990; Bonning et al.,J. Gen. Virol. 75:1551-6, 1994; and, Chazenbalk, and Rapoport, J. Biol.Chem. 270:1543-9, 1995. In such transfer vector constructs, a short orlong version of the basic protein promoter can be used. Moreover,transfer vectors can be constructed which replace the native zacrp2secretory signal sequences with secretory signal sequences derived frominsect proteins. For example, a secretory signal sequence fromEcdysteroid Glucosyltransferase (EGT), honey bee Melittin (Invitrogen,Carlsbad, Calif.), or baculovirus gp67 (PharMingen, San Diego, Calif.)can be used in constructs to replace the native zacrp2 secretory signalsequence. In addition, transfer vectors can include an in-frame fusionwith DNA encoding an epitope tag at the C- or N-terminus of theexpressed zacrp2 polypeptide, for example, a Glu-Glu epitope tag(Grussenmeyer et al., Proc. Natl. Acad. Sci. 82:7952-4, 1985). Using atechnique known in the art, a transfer vector containing zacrp2 istransformed into E. coli, and screened for bacmids which contain aninterrupted lacZ gene indicative of recombinant baculovirus. The bacmidDNA containing the recombinant baculovirus genome is isolated, usingcommon techniques, and used to transfect Spodoptera frugiperda cells,e.g. Sf9 cells. Recombinant virus that expresses zacrp2 is subsequentlyproduced. Recombinant viral stocks are made by methods commonly used theart.

The recombinant virus is used to infect host cells, typically a cellline derived from the fall armyworm, Spodoptera frugiperda. See, ingeneral, Glick and Pasternak, Molecular Biotechnology: Principles andApplications of Recombinant DNA, ASM Press, Washington, D.C., 1994.Another suitable cell line is the High FiveO™ cell line (Invitrogen)derived from Trichoplusia ni (U.S. Pat. No. 5,300,435). Commerciallyavailable serum-free media are used to grow and maintain the cells.Suitable media are Sf900 II™ (Life Technologies) or ESF 921™ (ExpressionSystems) for the Sf9 cells; and Ex-cellO405™ (JRH Biosciences, Lenexa,Kans.) or Express FiveO™ (Life Technologies) for the T. ni cells. Thecells are grown up from an inoculation density of approximately 2-5×10⁵cells to a density of 1-2×10⁶ cells at which time a recombinant viralstock is added at a multiplicity of infection (MOI) of 0.1 to 10, moretypically near 3. Procedures used are generally described in availablelaboratory manuals (King and Possee, ibid.; O'Reilly et al., ibid.;Richardson, ibid.). Subsequent purification of the zacrp2 polypeptidefrom the supernatant can be achieved using methods described herein.

Fungal cells, including yeast cells, can also be used within the presentinvention. Yeast species of particular interest in this regard includeSaccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica.Methods for transforming S. cerevisiae cells with exogenous DNA andproducing recombinant polypeptides therefrom are disclosed by, forexample, Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et al., U.S. Pat.No. 4,931,373; Brake, U.S. Pat. No. 4,870,008; Welch et al., U.S. Pat.No. 5,037,743; and Murray et al., U.S. Pat. No. 4,845,075. Transformedcells are selected by phenotype determined by the selectable marker,commonly drug resistance or the ability to grow in the absence of aparticular nutrient (e.g., leucine). A preferred vector system for usein Saccharomyces cerevisiae is the POT1 vector system disclosed byKawasaki et al. (U.S. Pat. No. 4,931,373), which allows transformedcells to be selected by growth in glucose-containing media. Suitablepromoters and terminators for use in yeast include those from glycolyticenzyme genes (see, e.g., Kawasaki, U.S. Pat. No. 4,599,311; Kingsman etal., U.S. Pat. No. 4,615,974; and Bitter, U.S. Pat. No. 4,977,092) andalcohol dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446;5,063,154; 5,139,936 and 4,661,454. Transformation systems for otheryeasts, including Hansenula polymorpha, Schizosaccharomyces pombe,Kluyveromryces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichiapastoris, Pichia methanolica, Pichia guillermondii and Candida maltosaare known in the art. See, for example, Gleeson et al., J. Gen.Microbiol. 132:3459-65, 1986 and Cregg, U.S. Pat. No. 4,882,279.Aspergillus cells may be utilized according to the methods of McKnightet al., U.S. Pat. No. 4,935,349. Methods for transforming Acremoniumchrysogenum are disclosed by Sumino et al., U.S. Pat. No. 5,162,228.Methods for transforming Neurospora are disclosed by Lambowitz, U.S.Pat. No. 4,486,533.

The use of Pichia methanolica as host for the production of recombinantproteins is disclosed in WIPO Publications WO 97/17450, WO 97/17451, WO98/02536, and WO 98/02565. DNA molecules for use in transforming P.methanolica will commonly be prepared as double-stranded, circularplasmids, which are preferably linearized prior to transformation. Forpolypeptide production in P. methanolica, it is preferred that thepromoter and terminator in the plasmid be that of a P. methanolica gene,such as a P. methanolica alcohol utilization gene (AUG1 or AUG2). Otheruseful promoters include those of the dihydroxyacetone synthase (DHAS),formate dehydrogenase (FMD), and catalase (CAT) genes. To facilitateintegration of the DNA into the host chromosome, it is preferred to havethe entire expression segment of the plasmid flanked at both ends byhost DNA sequences. A preferred selectable marker for use in Pichiamethanolica is a P. methanolica ADE2 gene, which encodesphosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21), whichallows ade2 host cells to grow in the absence of adenine. Forlarge-scale, industrial processes where it is desirable to minimize theuse of methanol, it is preferred to use host cells in which bothmethanol utilization genes (AUG1 and AUG2) are deleted. For productionof secreted proteins, host cells deficient in vacuolar protease genes(PEP4 and PRB1) are preferred. Electroporation is used to facilitate theintroduction of a plasmid containing DNA encoding a polypeptide ofinterest into P. methanolica cells. It is preferred to transform P.methanolica cells by electroporation using an exponentially decaying,pulsed electric field having a field strength of from 2.5 to 4.5 kV/cm,preferably about 3.75 kV/cm, and a time constant (T) of from 1 to 40milliseconds, most preferably about 20 milliseconds.

Prokaryotic host cells, including strains of the bacteria Escherichiacoli, Bacillus and other genera are also useful host cells within thepresent invention. Techniques for transforming these hosts andexpressing foreign DNA sequences cloned therein are well known in theart (see, e.g., Sambrook et al., ibid.). When expressing a zacrp2polypeptide in bacteria such as E. coli, the polypeptide may be retainedin the cytoplasm, typically as insoluble granules, or may be directed tothe periplasmic space by a bacterial secretion sequence. In the formercase, the cells are lysed, and the granules are recovered and denaturedusing, for example, guanidine isothiocyanate or urea. The denaturedpolypeptide can then be refolded and dimerized by diluting thedenaturant, such as by dialysis against a solution of urea and acombination of reduced and oxidized glutathione, followed by dialysisagainst a buffered saline solution. In the latter case, the polypeptidecan be recovered from the periplasmic space in a soluble and functionalform by disrupting the cells (by, for example, sonication or osmoticshock) to release the contents of the periplasmic space and recoveringthe protein, thereby obviating the need for denaturation and refolding.

Transformed or transfected host cells are cultured according toconventional procedures in a culture medium containing nutrients andother components required for the growth of the chosen host cells. Avariety of suitable media, including defined media and complex media,are known in the art and generally include a carbon source, a nitrogensource, essential amino acids, vitamins and minerals. Media may alsocontain such components as growth factors or serum, as required. Thegrowth medium will generally select for cells containing the exogenouslyadded DNA by, for example, drug selection or deficiency in an essentialnutrient which is complemented by the selectable marker carried on theexpression vector or co-transfected into the host cell.

Expressed recombinant zacrp2 polypeptides (or chimeric zacrp2polypeptides) can be purified using fractionation and/or conventionalpurification methods and media. Ammonium sulfate precipitation and acidor chaotrope extraction may be used for fractionation of samples.Exemplary purification steps may include hydroxyapatite, size exclusion,FPLC and reverse-phase high performance liquid chromatography. Suitablechromatographic media include derivatized dextrans, agarose, cellulose,polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Qderivatives are preferred. Exemplary chromatographic media include thosemedia derivatized with phenyl, butyl, or octyl groups, such asPhenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas,Montgomeryville, Pa.), Octyl-Sepharose (Pharmacia) and the like; orpolyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like.Suitable solid supports include glass beads, silica-based resins,cellulosic resins, agarose beads, cross-linked agarose beads,polystyrene beads, cross-linked polyacrylamide resins and the like thatare insoluble under the conditions in which they are to be used. Thesesupports may be modified with reactive groups that allow attachment ofproteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxylgroups and/or carbohydrate moieties. Examples of coupling chemistriesinclude cyanogen bromide activation, N-hydroxysuccinimide activation,epoxide activation, sulfhydryl activation, hydrazide activation, andcarboxyl and amino derivatives for carbodiimide coupling chemistries.These and other solid media are well known and widely used in the art,and are available from commercial suppliers. Methods for bindingreceptor polypeptides to support media are well known in the art.Selection of a particular method is a matter of routine design and isdetermined in part by the properties of the chosen support. See, forexample, Affinity Chromatography: Principles & Methods, Pharmacia LKBBiotechnology, Uppsala, Sweden, 1988.

The polypeptides of the present invention can be isolated byexploitation of their structural or binding properties. For example,immobilized metal ion adsorption (IMAC) chromatography can be used topurify histidine-rich proteins or proteins having a His tag. Briefly, agel is first charged with divalent metal ions to form a chelate(Sulkowski, Trends in Biochem. 3:1-7, 1985). Histidine-rich proteinswill be adsorbed to this matrix with differing affinities, dependingupon the metal ion used, and will be eluted by competitive elution,lowering the pH, or use of strong chelating agents. Other methods ofpurification include purification of glycosylated proteins by lectinaffinity chromatography and ion exchange chromatography (Methods inEnzymol., Vol. 182, “Guide to Protein Purification”, M. Deutscher,(ed.), Acad. Press, San Diego, 1990, pp. 529-39). Within an additionalpreferred embodiments of the invention, a fusion of the polypeptide ofinterest and an affinity tag (e.g., maltose-binding protein, FLAG,Glu-Glu, an immunoglobulin domain) may be constructed to facilitatepurification as is discussed in greater detail in the Example sectionsbelow.

Protein refolding (and optionally, reoxidation) procedures may beadvantageously used. It is preferred to purify the protein to >80%purity, more preferably to >90% purity, even more preferably >95%, andparticularly preferred is a pharmaceutically pure state, that is greaterthan 99.9% pure with respect to contaminating macromolecules,particularly other proteins and nucleic acids, and free of infectiousand pyrogenic agents. Preferably, a purified protein is substantiallyfree of other proteins, particularly other proteins of animal origin.

Zacrp2 polypeptides or fragments thereof may also be prepared throughchemical synthesis by methods well known in the art. Such zacrp2polypeptides may be monomers or multimers; glycosylated ornon-glycosylated; pegylated or non-pegylated; and may or may not includean initial methionine amino acid residue.

A ligand-binding polypeptide, such as a zacrp2-binding polypeptide, canalso be used for purification of ligand. The polypeptide is immobilizedon a solid support, such as beads of agarose, cross-linked agarose,glass, cellulosic resins, silica-based resins, polystyrene, cross-linkedpolyacrylamide, or like materials that are stable under the conditionsof use. Methods for linking polypeptides to solid supports are known inthe art, and include amine chemistry, cyanogen bromide activation,N-hydroxysuccinimide activation, epoxide activation, sulfhydrylactivation, and hydrazide activation. The resulting medium willgenerally be configured in the form of a column, and fluids containingligand are passed through the column one or more times to allow ligandto bind to the ligand-binding polypeptide. The ligand is then elutedusing changes in salt concentration, chaotropic agents (guanidine HCl),or pH to disrupt ligand-receptor binding.

An assay system that uses a ligand-binding receptor (or an antibody, onemember of a complement/anti-complement pair) or a binding fragmentthereof, and a commercially available biosensor instrument (BIAcore™,Pharmacia Biosensor, Piscataway, N.J.) may be advantageously employed.Such receptor, antibody, member of a complement/anti-complement pair orfragment is immobilized onto the surface of a receptor chip. Use of thisinstrument is disclosed by Karlsson, J. Immunol. Methods 145:229-40,1991 and Cunningham and Wells, J. Mol. Biol. 234:554-63, 1993. Areceptor, antibody, member or fragment is covalently attached, usingamine or sulfhydryl chemistry, to dextran fibers that are attached togold film within the flow cell. A test sample is passed through thecell. If a ligand, epitope, or opposite member of thecomplement/anti-complement pair is present in the sample, it will bindto the immobilized receptor, antibody or member, respectively, causing achange in the refractive index of the medium, which is detected as achange in surface plasmon resonance of the gold film. This system allowsthe determination of on- and off-rates, from which binding affinity canbe calculated, and assessment of stoichiometry of binding.

Ligand-binding polypeptides can also be used within other assay systemsknown in the art. Such systems include Scatchard analysis fordetermination of binding affinity (see Scatchard, Ann. NY Acad. Sci. 51:660-72, 1949) and calorimetric assays (Cunningham et al., Science253:545-48, 1991; Cunningham et al., Science 245:821-25, 1991).

The invention also provides anti-zacrp2 antibodies. Antibodies to zacrp2can be obtained, for example, using as an antigen the product of azacrp2 expression vector, or zacrp2 isolated from a natural source.Particularly useful anti-zacrp2 antibodies “bind specifically” withzacrp2. Antibodies are considered to be specifically binding if theantibodies bind to a zacrp2 polypeptide, peptide or epitope with abinding affinity (K_(a)) of 10⁶ M⁻¹ or greater, preferably 10⁷ M⁻¹ orgreater, more preferably 10⁸ M⁻¹ or greater, and most preferably 10⁹ M⁻¹or greater. The binding affinity of an antibody can be readilydetermined by one of ordinary skill in the art, for example, byScatchard analysis (Scatchard, Ann. NY Acad. Sci. 51:660, 1949).Suitable antibodies include antibodies that bind with zacrp2 inparticular domains.

Anti-zacrp2 antibodies can be produced using antigenic zacrp2epitope-bearing peptides and polypeptides. Antigenic epitope-bearingpeptides and polypeptides of the present invention contain a sequence ofat least nine, preferably between 15 to about 30 amino acids containedwithin SEQ ID NO:2. However, peptides or polypeptides comprising alarger portion of an amino acid sequence of the invention, containingfrom 30 to 50 amino acids, or any length up to and including the entireamino acid sequence of a polypeptide of the invention, also are usefulfor inducing antibodies that bind with zacrp2. It is desirable that theamino acid sequence of the epitope-bearing peptide is selected toprovide substantial solubility in aqueous solvents (i.e., the sequenceincludes relatively hydrophilic residues, while hydrophobic residues arepreferably avoided). Hydrophilic peptides can be predicted by one ofskill in the art from a hydrophobicity plot, see for example, Hopp andWoods (Proc. Nat. Acad. Sci. USA 78:3824-8, 1981) and Kyte and Doolittle(J. Mol. Biol. 157: 105-142, 1982). Moreover, amino acid sequencescontaining proline residues may be also be desirable for antibodyproduction.

Polyclonal antibodies to recombinant zacrp2 protein or to zacrp2isolated from natural sources can be prepared using methods well-knownto those of skill in the art. See, for example, Green et al.,“Production of Polyclonal Antisera,” in Immunochemical Protocols(Manson, ed.), pages 1-5 (Humana Press 1992), and Williams et al.,“Expression of foreign proteins in E. coli using plasmid vectors andpurification of specific polyclonal antibodies,” in DNA Cloning 2:Expression Systems, 2nd Edition, Glover et al. (eds.), page 15 (OxfordUniversity Press 1995). The immunogenicity of a zacrp2 polypeptide canbe increased through the use of an adjuvant, such as alum (aluminumhydroxide) or Freund's complete or incomplete adjuvant. Polypeptidesuseful for immunization also include fusion polypeptides, such asfusions of zacrp2 or a portion thereof with an immunoglobulinpolypeptide or with maltose binding protein. The polypeptide immunogenmay be a full-length molecule or a portion thereof. If the polypeptideportion is “hapten-like,” such portion may be advantageously joined orlinked to a macromolecular carrier (such as keyhole limpet hemocyanin(KLH), bovine serum albumin (BSA) or tetanus toxoid) for immunization.

Although polyclonal antibodies are typically raised in animals such ashorses, cows, dogs, chicken, rats, mice, rabbits, hamsters, guinea pigs,goats, or sheep, an anti-zacrp2 antibody of the present invention mayalso be derived from a subhuman primate antibody. General techniques forraising diagnostically and therapeutically useful antibodies in baboonsmay be found, for example, in Goldenberg et al., international patentpublication No. WO 91/11465, and in Losman et al., Int. J. Cancer46:310, 1990. Antibodies can also be raised in transgenic animals suchas transgenic sheep, cows, goats or pigs, and can also be expressed inyeast and fungi in modified forms as will as in mammalian and insectcells.

Alternatively, monoclonal anti-zacrp2 antibodies can be generated.Rodent monoclonal antibodies to specific antigens may be obtained bymethods known to those skilled in the art (see, for example, Kohler etal., Nature 256:495 (1975), Coligan et al. (eds.), Current Protocols inImmunology, Vol. 1, pages 2.5.1-2.6.7 (John Wiley & Sons 1991), Picksleyet al., “Production of monoclonal antibodies against proteins expressedin E. coli,” in DNA Cloning 2: Expression Systems, 2nd Edition, Gloveret al. (eds.), page 93 (Oxford University Press 1995)).

Briefly, monoclonal antibodies can be obtained by injecting mice with acomposition comprising a zacrp2 gene product, verifying the presence ofantibody production by removing a serum sample, removing the spleen toobtain B-lymphocytes, fusing the B-lymphocytes with myeloma cells toproduce hybridomas, cloning the hybridomas, selecting positive cloneswhich produce antibodies to the antigen, culturing the clones thatproduce antibodies to the antigen, and isolating the antibodies from thehybridoma cultures.

In addition, an anti-zacrp2 antibody of the present invention may bederived from a human monoclonal antibody. Human monoclonal antibodiesare obtained from transgenic mice that have been engineered to producespecific human antibodies in response to antigenic challenge. In thistechnique, elements of the human heavy and light chain locus areintroduced into strains of mice derived from embryonic stem cell linesthat contain targeted disruptions of the endogenous heavy chain andlight chain loci. The transgenic mice can synthesize human antibodiesspecific for human antigens, and the mice can be used to produce humanantibody-secreting hybridomas. Methods for obtaining human antibodiesfrom transgenic mice are described, for example, by Green et al., NatureGenet. 7:13, 1994, Lonberg et al., Nature 368:856, 1994, and Taylor etal., Int. Immun. 6:579, 1994.

Monoclonal antibodies can be isolated and purified from hybridomacultures by a variety of well-established techniques. Such isolationtechniques include affinity chromatography with Protein-A Sepharose,size-exclusion chromatography, and ion-exchange chromatography (see, forexample, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3; Baines etal., “Purification of Immunoglobulin G (IgG),” in Methods in MolecularBiology, Vol. 10, pages 79-104 (The Humana Press, Inc. 1992)).

For particular uses, it may be desirable to prepare fragments ofanti-zacrp2 antibodies. Such antibody fragments can be obtained, forexample, by proteolytic hydrolysis of the antibody. Antibody fragmentscan be obtained by pepsin or papain digestion of whole antibodies byconventional methods. As an illustration, antibody fragments can beproduced by enzymatic cleavage of antibodies with pepsin to provide a 5Sfragment denoted F(ab′)₂. This fragment can be further cleaved using athiol reducing agent to produce 3.5S Fab′ monovalent fragments.Optionally, the cleavage reaction can be performed using a blockinggroup for the sulfhydryl groups that result from cleavage of disulfidelinkages. As an alternative, an enzymatic cleavage using pepsin producestwo monovalent Fab fragments and an Fc fragment directly. These methodsare described, for example, by Goldenberg, U.S. Pat. No. 4,331,647,Nisonoff et al., Arch Biochem. Biophys. 89:230, 1960, Porter, Biochem.J. 73:119, 1959, Edelman et al., in Methods in Enzymology Vol. 1, page422 (Academic Press 1967), and by Coligan, ibid.

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

For example, Fv fragments comprise an association of V_(H) and V_(L)chains. This association can be noncovalent, as described by Inbar etal., Proc. Natl. Acad. Sci. USA 69:2659, 1972. Alternatively, thevariable chains can be linked by an intermolecular disulfide bond orcross-linked by chemicals such as gluteraldehyde (see, for example,Sandhu, Crit. Rev. Biotech. 12:437, 1992).

The Fv fragments may comprise V_(H) and V_(L) chains which are connectedby a peptide linker. These single-chain antigen binding proteins (scFv)are prepared by constructing a structural gene comprising DNA sequencesencoding the V_(H) and V_(L) domains which are connected by anoligonucleotide. The structural gene is inserted into an expressionvector which is subsequently introduced into a host cell, such as E.coli. The recombinant host cells synthesize a single polypeptide chainwith a linker peptide bridging the two V domains. Methods for producingscFvs are described, for example, by Whitlow et al., Methods: ACompanion to Methods in Enzymology 2:97, 1991, also see, Bird et al.,Science 242:423, 1988, Ladner et al., U.S. Pat. No. 4,946,778, Pack etal., Bio/Technology 11:1271, 1993, and Sandhu, ibid.

As an illustration, a scFV can be obtained by exposing lymphocytes tozacrp2 polypeptide in vitro, and selecting antibody display libraries inphage or similar vectors (for instance, through use of immobilized orlabeled zacrp2 protein or peptide). Genes encoding polypeptides havingpotential zacrp2 polypeptide binding domains can be obtained byscreening random peptide libraries displayed on phage (phage display) oron bacteria, such as E. coli. Nucleotide sequences encoding thepolypeptides can be obtained in a number of ways, such as through randommutagenesis and random polynucleotide synthesis. These random peptidedisplay libraries can be used to screen for peptides which interact witha known target which can be a protein or polypeptide, such as a ligandor receptor, a biological or synthetic macromolecule, or organic orinorganic substances. Techniques for creating and screening such randompeptide display libraries are known in the art (Ladner et al., U.S. Pat.No. 5,223,409, Ladner et al., U.S. Pat. No. 4,946,778, Ladner et al.,U.S. Pat. No. 5,403,484, Ladner et al., U.S. Pat. No. 5,571,698, and Kayet al., Phage Display of Peptides and Proteins (Academic Press, Inc.1996)) and random peptide display libraries and kits for screening suchlibraries are available commercially, for instance from Clontech (PaloAlto, Calif.), Invitrogen Inc. (San Diego, Calif.), New England Biolabs,Inc. (Beverly, Mass.), and Pharmacia LKB Biotechnology Inc. (Piscataway,N.J.). Random peptide display libraries can be screened using the zacrp2sequences disclosed herein to identify proteins which bind to zacrp2.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells (see, for example, Larrick et al.,Methods: A Companion to Methods in Enzymology 2:106, 1991),Courtenay-Luck, “Genetic Manipulation of Monoclonal Antibodies,” inMonoclonal Antibodies: Production, Engineering and Clinical Application,Ritter et al. (eds.), page 166 (Cambridge University Press, 1995), andWard et al., “Genetic Manipulation and Expression of Antibodies,” inMonoclonal Antibodies: Principles and Applications, Birch et al.,(eds.), page 137 (Wiley-Liss, Inc. 1995)).

Alternatively, an anti-zacrp2 antibody may be derived from a “humanized”monoclonal antibody. Humanized monoclonal antibodies are produced bytransferring mouse complementary determining regions from heavy andlight variable chains of the mouse immunoglobulin into a human variabledomain. Typical residues of human antibodies are then substituted in theframework regions of the murine counterparts. The use of antibodycomponents derived from humanized monoclonal antibodies obviatespotential problems associated with the immunogenicity of murine constantregions. General techniques for cloning murine immunoglobulin variabledomains are described, for example, by Orlandi et al., Proc. Nat. Acad.Sci. USA 86:3833, 1989. Techniques for producing humanized monoclonalantibodies are described, for example, by Jones et al., Nature 321:522,1986, Carter et al., Proc. Nat. Acad. Sci. USA 89:4285, 1992, Sandhu,Crit. Rev. Biotech. 12:437, 1992, Singer et al., J. Immun. 150:2844,1993, Sudhir (ed.), Antibody Engineering Protocols (Humana Press, Inc.1995), Kelley, “Engineering Therapeutic Antibodies,” in ProteinEngineering: Principles and Practice, Cleland et al. (eds.), pages399-434 (John Wiley & Sons, Inc. 1996), and by Queen et al., U.S. Pat.No. 5,693,762 (1997).

Polyclonal anti-idiotype antibodies can be prepared by immunizinganimals with anti-zacrp2 antibodies or antibody fragments, usingstandard techniques. See, for example, Green et al., “Production ofPolyclonal Antisera,” in Methods In Molecular Biology: ImmunochemicalProtocols, Manson (ed.), pages 1-12 (Humana Press 1992). Also, seeColigan, ibid. at pages 2.4.1-2.4.7. Alternatively, monoclonalanti-idiotype antibodies can be prepared using anti-zacrp2 antibodies orantibody fragments as immunogens with the techniques, described above.As another alternative, humanized anti-idiotype antibodies or subhumanprimate anti-idiotype antibodies can be prepared using theabove-described techniques. Methods for producing anti-idiotypeantibodies are described, for example, by Irie, U.S. Pat. No. 5,208,146,Greene, et. al., U.S. Pat. No. 5,637,677, and Varthakavi and Minocha, J.Gen. Virol. 77:1875, 1996.

Genes encoding polypeptides having potential zacrp2 polypeptide bindingdomains, “binding proteins”, can be obtained by screening random ordirected peptide libraries displayed on phage (phage display) or onbacteria, such as E. coli. Nucleotide sequences encoding thepolypeptides can be obtained in a number of ways, such as through randommutagenesis and random polynucleotide synthesis. Alternatively,constrained phage display libraries can also be produced. These peptidedisplay libraries can be used to screen for peptides which interact witha known target which can be a protein or polypeptide, such as a ligandor receptor, a biological or synthetic macromolecule, or organic orinorganic substances. Techniques for creating and screening such peptidedisplay libraries are known in the art (Ladner et al., U.S. Pat. No.5,223,409; Ladner et al., U.S. Pat. No. 4,946,778; Ladner et al., U.S.Pat. No. 5,403,484 and Ladner et al., U.S. Pat. No. 5,571,698) andpeptide display libraries and kits for screening such libraries areavailable commercially, for instance from Clontech (Palo Alto, Calif.),Invitrogen Inc. (San Diego, Calif.), New England Biolabs, Inc. (Beverly,Mass.) and Pharmacia LKB Biotechnology Inc. (Piscataway, N.J.). Peptidedisplay libraries can be screened using the zacrp2 sequences disclosedherein to identify proteins which bind to zacrp2. These “bindingproteins” which interact with zacrp2 polypeptides can be usedessentially like an antibody.

A variety of assays known to those skilled in the art can be utilized todetect antibodies and/or binding proteins which specifically bind tozacrp2 proteins or peptides. Exemplary assays are described in detail inAntibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold SpringHarbor Laboratory Press, 1988. Representative examples of such assaysinclude: concurrent immunoelectrophoresis, radioimmunoassay,radioimmuno-precipitation, enzyme-linked immunosorbent assay (ELISA),dot blot or Western blot assay, inhibition or competition assay, andsandwich assay. In addition, antibodies can be screened for binding towild-type versus mutant zacrp2 protein or polypeptide.

Antibodies and binding proteins to zacrp2 may be used for tagging cellsthat express zacrp2; for isolating zacrp2 by affinity purification; fordiagnostic assays for determining circulating levels of zacrp2polypeptides; for detecting or quantitating soluble zacrp2 as marker ofunderlying pathology or disease; in analytical methods employing FACS;for screening expression libraries; for generating anti-idiotypicantibodies; and as neutralizing antibodies or as antagonists to blockzacrp2 polypeptide modulation of spermatogenesis or like activity invitro and in vivo. Suitable direct tags or labels include radionuclides,enzymes, substrates, cofactors, inhibitors, fluorescent markers,chemiluminescent markers, magnetic particles and the like; indirect tagsor labels may feature use of biotin-avidin or othercomplement/anti-complement pairs as intermediates. Moreover, antibodiesto zacrp2 or fragments thereof may be used in vitro to detect denaturedzacrp2 or fragments thereof in assays, for example, Western Blots orother assays known in the art.

Antibodies or polypeptides herein can also be directly or indirectlyconjugated to drugs, toxins, radionuclides and the like, and theseconjugates used for in vivo diagnostic or therapeutic applications. Forinstance, polypeptides or antibodies of the present invention can beused to identify or treat tissues or organs that express a correspondinganti-complementary molecule (receptor or antigen, respectively, forinstance). More specifically, zacrp2 polypeptides or anti-zacrp2antibodies, or bioactive fragments or portions thereof, can be coupledto detectable or cytotoxic molecules and delivered to a mammal havingcells, tissues or organs that express the anti-complementary molecule.

An additional aspect of the present invention provides methods foridentifying agonists or antagonists of the zacrp2 polypeptides disclosedabove, which agonists or antagonists may have valuable properties asdiscussed further herein. Within one embodiment, there is provided amethod of identifying zacrp2 polypeptide agonists, comprising providingcells responsive thereto, culturing the cells in the presence of a testcompound and comparing the cellular response with the cell cultured inthe presence of the zacrp2 polypeptide, and selecting the test compoundsfor which the cellular response is of the same type.

Within another embodiment, there is provided a method of identifyingantagonists of zacrp2 polypeptide, comprising providing cells responsiveto a zacrp2 polypeptide, culturing a first portion of the cells in thepresence of zacrp2 polypeptide, culturing a second portion of the cellsin the presence of the zacrp2 polypeptide and a test compound, anddetecting a decrease in a cellular response of the second portion of thecells as compared to the first portion of the cells. In addition tothose assays disclosed herein, samples can be tested for inhibition ofzacrp2 activity within a variety of assays designed to measure receptorbinding or the stimulation/inhibition of zacrp2-dependent cellularresponses. For example, zacrp2-responsive cell lines can be transfectedwith a reporter gene construct that is responsive to a zacrp2-stimulatedcellular pathway. Reporter gene constructs of this type are known in theart, and will generally comprise a zacrp2-DNA response element operablylinked to a gene encoding an assayable protein, such as luciferase. DNAresponse elements can include, but are not limited to, cyclic AMPresponse elements (CRE), hormone response elements (HRE), insulinresponse element (IRE) (Nasrin et al., Proc. Natl. Acad. Sci. USA87:5273-7, 1990) and serum response elements (SRE) (Shaw et al. Cell 56:563-72, 1989). Cyclic AMP response elements are reviewed in Roestler etal., J. Biol. Chem. 263 (19):9063-6, 1988 and Habener, Molec.Endocrinol. 4 (8):1087-94, 1990. Hormone response elements are reviewedin Beato, Cell 56:335-44; 1989. Candidate compounds, solutions, mixturesor extracts are tested for the ability to inhibit the activity of zacrp2on the target cells as evidenced by a decrease in zacrp2 stimulation ofreporter gene expression. Assays of this type will detect compounds thatdirectly block zacrp2 binding to cell-surface receptors, as well ascompounds that block processes in the cellular pathway subsequent toreceptor-ligand binding. In the alternative, compounds or other samplescan be tested for direct blocking of zacrp2 binding to receptor usingzacrp2 tagged with a detectable label (e.g., ¹²⁵I, biotin, horseradishperoxidase, FITC, and the like). Within assays of this type, the abilityof a test sample to inhibit the binding of labeled zacrp2 to thereceptor is indicative of inhibitory activity, which can be confirmedthrough secondary assays. Receptors used within binding assays may becellular receptors or isolated, immobilized receptors.

Zacrp2 polypeptides, fragments, fusions, agonists or antagonists can beused to modulate energy balance in mammals or to protect endothelialcells from injury. With regard to modulating energy balance, zacrp2polypeptides modulate cellular metabolic reactions. Such metabolicreactions include adipogenesis, gluconeogenesis, glycogenolysis,lipogenesis, glucose uptake, protein synthesis, thermogenesis, oxygenutilization and the like. The expression pattern of zacrp2 polypeptideindicates expression in endothelial cell tissues. With regard toendothelial cell protection, zacrp2 polypeptide may be used in organpreservation, for cryopreservation, for surgical pretreatment to preventinjury due to ischemia and/or inflammation or in like procedures.Expression of zacrp2 polypeptide in the heart suggests that the proteinmay modulate acetylcholine and/or norepinephrine release. Zacrp2polypeptides may also find use as neurotransmitters or as modulators ofneurotransmission, as indicated by expression of the polypeptide intissues associated with the sympathetic or parasympathetic nervoussystem. In this regard, zacrp2 polypeptides may find utility inmodulating nutrient uptake, as demonstrated, for example, by2-deoxy-glucose uptake in the brain or the like.

Expression in the aorta suggests that the protein may be involved inhemostasis. Platelets interact with damaged vessel walls to form athrombus. The degree of response is graded due to the subendotheliumtissue exposed and the blood flow in the injured area. In this regard,zacrp2 polypeptides may find utility in modulating hemostasis,increasing blood flow flowing vascular injury and pacifying collagenoussurfaces.

Among other methods known in the art or described herein, mammalianenergy balance may be evaluated by monitoring one or more of thefollowing metabolic functions: adipogenesis, gluconeogenesis,glycogenolysis, lipogenesis, glucose uptake, protein synthesis,thermogenesis, oxygen utilization or the like. These metabolic functionsare monitored by techniques (assays or animal models) known to one ofordinary skill in the art, as is more fully set forth below. Forexample, the glucoregulatory effects of insulin are predominantlyexerted in the liver, skeletal muscle and adipose tissue. Insulin bindsto its cellular receptor in these three tissues and initiatestissue-specific actions that result in, for example, the inhibition ofglucose production and the stimulation of glucose utilization. In theliver, insulin stimulates glucose uptake and inhibits gluconeogenesisand glycogenolysis. In skeletal muscle and adipose tissue, insulin actsto stimulate the uptake, storage and utilization of glucose.

Art-recognized methods exist for monitoring all of the metabolicfunctions recited above. Thus, one of ordinary skill in the art is ableto evaluate zacrp2 polypeptides, fragments, fusion proteins, antibodies,agonists and antagonists for metabolic modulating functions. Exemplarymodulating techniques are set forth below.

Adipogenesis, gluconeogenesis and glycogenolysis are interrelatedcomponents of mammalian energy balance, which may be evaluated by knowntechniques using, for example, ob/ob mice or db/db mice. The ob/ob miceare inbred mice that are homozygous for an inactivating mutation at theob (obese) locus. Such ob/ob mice are hyperphagic and hypometabolic, andare believed to be deficient in production of circulating OB protein.The db/db mice are inbred mice that are homozygous for an inactivatingmutation at the db (diabetes) locus. The db/db mice display a phenotypesimilar to that of ob/ob mice, except db/db mice also display a diabeticphenotype. Such db/db mice are believed to be resistant to the effectsof circulating OB protein. Also, various in vitro methods of assessingthese parameters are known in the art.

Insulin-stimulated lipogenesis, for example, may be monitored bymeasuring the incorporation of ¹⁴C-acetate into triglyceride (Mackall etal. J. Biol. Chem. 251:6462-4, 1976) or triglyceride accumulation(Kletzien et al., Mol. Pharmacol. 41:393-8, 1992).

Glucose uptake may be evaluated, for example, in an assay forinsulin-stimulated glucose transport. Non-transfected, differentiated L6myotubes (maintained in the absence of G418) are placed in DMEMcontaining 1 g/l glucose, 0.5 or 1.0% BSA, 20 mM Hepes, and 2 mMglutamine. After two to five hours of culture, the medium is replacedwith fresh, glucose-free DMEM containing 0.5 or 1.0% BSA, 20 mM Hepes, 1mM pyruvate, and 2 mM glutamine. Appropriate concentrations of insulinor IGF-1, or a dilution series of the test substance, are added, and thecells are incubated for 20-30 minutes. ³H or ¹⁴C-labeled deoxyglucose isadded to ≈50 lM final concentration, and the cells are incubated forapproximately 10-30 minutes. The cells are then quickly rinsed with coldbuffer (e.g. PBS), then lysed with a suitable lysing agent (e.g. 1% SDSor 1 N NaOH). The cell lysate is then evaluated by counting in ascintillation counter. Cell-associated radioactivity is taken as ameasure of glucose transport after subtracting non-specific binding asdetermined by incubating cells in the presence of cytocholasin b, aninhibitor of glucose transport. Other methods include those describedby, for example, Manchester et al., Am. J. Physiol. 266 (Endocrinol.Metab. 29):E326-E333, 1994 (insulin-stimulated glucose transport).

Protein synthesis may be evaluated, for example, by comparingprecipitation of ³⁵S-methionine-labeled proteins following incubation ofthe test cells with ³⁵S-methionine and ³⁵S-methionine and a putativemodulator of protein synthesis.

Thermogenesis may be evaluated as described by B. Stanley in The Biologyof Neuropeptide Y and Related Peptides, W. Colmers and C. Wahlestedt(eds.), Humana Press, Ottawa, 1993, pp. 457-509; C. Billington et al.,Am. J. Physiol. 260:R321, 1991; N. Zarjevski et al., Endocrinology133:1753, 1993; C. Billington et al., Am. J. Physiol. 266:R1765, 1994;Heller et al., Am. J. Physiol. 252(4 Pt 2): R661-7, 1987; and Heller etal., Am. J. Physiol. 245: R321-8, 1983. Also, metabolic rate, which maybe measured by a variety of techniques, is an indirect measurement ofthermogenesis.

Oxygen utilization may be evaluated as described by Heller et al.,Pflugers Arch 369: 55-9, 1977. This method also involved an analysis ofhypothalmic temperature and metabolic heat production. Oxygenutilization and thermoregulation have also been evaluated in humans asdescribed by Haskell et al., J. Appl. Physiol. 51: 948-54, 1981.

Among other methods known in the art or described herein, mammalianendothelial cell tissue protection may be evaluated by monitoring thefunction of endothelial tissue. For example, the function of the heart(aorta) may be evaluated by monitoring acetylcholine release,norepinephrine release or like parameters. These parameters aremonitored by techniques (assays or animal models) known to one ofordinary skill in the art, as is more fully set forth below.

Acetylcholine and norepinephrine release may be monitored by HPLC. Levy,Electrophysiology of the Sinoatrial and Atrioventricular Nodes, Alan R.Liss, Inc., 187-197, 1998, describe measurement of norepinephrine incoronary sinus effluent. In addition, animals may be electrically paced,with the results monitored as described by Elsner, European HeartJournal 16(Supplement N) 52-8, 1995, and Reiffel and Kuehnert, PACE17(Part 1): 349-65, 1994.

Among other methods known in the art or described herein,neurotransmission functions may be evaluated by monitoring2-deoxy-glucose uptake in the brain. This parameter is monitored bytechniques (assays or animal models) known to one of ordinary skill inthe art, for example, autoradiography. Useful monitoring techniques aredescribed, for example, by Kilduff et al., J. Neurosci. 10 2463-75,1990, with related techniques used to evaluate the “hibernating heart”as described in Gerber et al. Circulation 94: 651-8, 1996, andFallavollita et al., Circulation 95: 1900-9, 1997.

In addition, zacrp2 polypeptides, fragments, fusions agonists orantagonists thereof may be therapeutically useful for anti-microbialapplications. For example, complement component C1q plays a role in hostdefense against infectious agents, such as bacteria and viruses. C1q isknown to exhibit several specialized functions. For example, C1qtriggers the complement cascade via interaction with bound antibody orC-reactive protein (CRP). Also, C1q interacts directly with certainbacteria, RNA viruses, mycoplasma, uric acid crystals, the lipid Acomponent of bacterial endotoxin and membranes of certain intracellularorganelles. C1q binding to the C1q receptor is believed to promotephagocytosis. C1q also appears to enhance the antibody formation aspectof the host defense system. See, for example, Johnston, Pediatr. Infect.Dis. J. 12(11): 933-41, 1993. Thus, soluble C1q-like molecules may beuseful as anti-microbial agents, promoting lysis or phagocytosis ofinfectious agents.

Zacrp2 fragments as well as zacrp2 polypeptides, fusion proteins,agonists, antagonists or antibodies may be evaluated with respect totheir anti-microbial properties according to procedures known in theart. See, for example, Barsum et al., Eur. Respir. J. 8(5): 709-14,1995; Sandovsky-Losica et al., J. Med. Vet. Mycol (England) 28(4):279-87, 1990; Mehentee et al., J. Gen. Microbiol. (England) 135 (Pt. 8):2181-8, 1989; Segal and Savage, J. Med. Vet. Mycol. 24: 477-9, 1986 andthe like. If desired, the performance of zacrp2 in this regard can becompared to proteins known to be functional in this regard, such asproline-rich proteins, lysozyme, histatins, lactoperoxidase or the like.In addition, zacrp2 fragments, polypeptides, fusion proteins, agonists,antagonists or antibodies may be evaluated in combination with one ormore anti-microbial agents to identify synergistic effects. One ofordinary skill in the art will recognize that the anti-microbialproperties of zacrp2 polypeptides, fragments, fusion proteins, agonists,antagonists and antibodies may be similarly evaluated.

As neurotransmitters or neurotransmission modulators, zacrp2 polypeptidefragments as well as zacrp2 polypeptides, fusion proteins, agonists,antagonists or antibodies of the present invention may also modulatecalcium ion concentration, muscle contraction, hormone secretion, DNAsynthesis or cell growth, inositol phosphate turnover, arachidonaterelease, phospholipase-C activation, gastric emptying, human neutrophilactivation or ADCC capability, superoxide anion production and the like.Evaluation of these properties can be conducted by known methods, suchas those set forth herein.

The impact of zacrp2 polypeptide, fragment, fusion, antibody, agonist orantagonist on intracellular calcium level may be assessed by methodsknown in the art, such as those described by Dobrzanski et al.,Regulatory Peptides 45: 341-52, 1993, and the like. The impact of zacrp2polypeptide, fragment, fusion, agonist or antagonist on musclecontraction may be assessed by methods known in the art, such as thosedescribed by Smits & Lebebvre, J. Auton. Pharmacol. 14: 383-92, 1994,Belloli et al., J. Vet. Pharmacol. Therap. 17: 379-83, 1994, Maggi etal., Regulatory Peptides 53: 259-74, 1994, and the like. The impact ofzacrp2 polypeptide, fragment, fusion, agonist or antagonist on hormonesecretion may be assessed by methods known in the art, such as those forprolactin release described by Henriksen et al., J. Recep. Sig. Transd.Res. 15(1-4): 529-41, 1995, and the like. The impact of zacrp2polypeptide, fragment, fusion, agonist or antagonist on DNA synthesis orcell growth may be assessed by methods known in the art, such as thosedescribed by Dobrzanski et al., Regulatory Peptides 45: 341-52, 1993,and the like. The impact of zacrp2 polypeptide, fragment, fusion,agonist or antagonist on inositol phosphate turnover may be assessed bymethods known in the art, such as those described by Dobrzanski et al.,Regulatory Peptides 45: 341-52, 1993, and the like.

Also, the impact of zacrp2 polypeptide, fragment, fusion, agonist orantagonist on arachidonate release may be assessed by methods known inthe art, such as those described by Dobrzanski et al., RegulatoryPeptides 45: 341-52, 1993, and the like. The impact of zacrp2polypeptide, fragment, fusion, agonist or antagonist on phospholipase-Cactivation may be assessed by methods known in the art, such as thosedescribed by Dobrzanski et al., Regulatory Peptides 45: 341-52, 1993,and the like. The impact of zacrp2 polypeptide, fragment, fusion,agonist or antagonist on gastric emptying may be assessed by methodsknown in the art, such as those described by Varga et al., Eur. J.Pharmacol. 286: 109-112, 1995, and the like. The impact of zacrp2polypeptide, fragment, fusion, agonist or antagonist on human neutrophilactivation and ADCC capability may be assessed by methods known in theart, such as those described by Wozniak et al., Immunology 78: 629-34,1993, and the like. The impact of zacrp2 polypeptide, fragment, fusion,agonist or antagonist on superoxide anion production may be assessed bymethods known in the art, such as those described by Wozniak et al.,Immunology 78: 629-34, 1993, and the like.

Collagen is a potent inducer of platelet aggregation. This poses risksto patients recovering from vascular injures. Inhibitors ofcollagen-induced platelet aggregation would be useful for blocking thebinding of platelets to collagen-coated surfaces and reducing associatedcollagen-induced platelet aggregation. C1q is a component of thecomplement pathway and has been found to stimulate defense mechanisms aswell as trigger the generation of toxic oxygen species that can causetissue damage (Tenner, Behring Inst. Mitt. 93:241-53, 1993). C1q bindingsites are found on platelets. C1q, independent of an immune bindingpartner, has been found to inhibit platelet aggregation but not plateletadhesion or shape change. The amino terminal region of C1q shareshomology with collagen (Peerschke and Ghebrehiwet, J. Immunol.145:2984-88, 1990). Inhibition of C1q and the complement pathway can bedetermined using methods disclosed herein or know in the art, such asdescribed in Suba and Csako, J. Immunol. 117:304-9, 1976.

The impact of zacrp2 polypeptide, fragments, fusions, agonists orantagonists on collagen-mediated platelet adhesion, activation andaggregation may be evaluated using methods described herein or known inthe art, such as the platelet aggregation assay (Chiang et al.,Thrombosis Res. 37:605-12, 1985) and platelet adhesion assays (Peerschkeand Ghebrehiwet, J. Immunol. 144:221-25, 1990). Assays for plateletadhesion to collagen and inhibition of collagen-induced plateletaggregation can be measured using methods described in Keller et al., J.Biol. Chem. 268:5450-6, 1993; Waxman and Connolly, J. Biol. Chem.268:5445-9, 1993; Noeske-Jungblut et al., J. Biol. Chem. 269:5050-3 or1994 Deckmyn et al., Blood 85:712-9, 1995.

The impact of zacrp2 polypeptide, fragments, fusions, agonists orantagonists on vasodilation of aortic rings can be measured according tothe methods of Dainty et al., J. Pharmacol. 100:767, 1990 and Rhee etal., Neurotox. 16:179, 1995.

Various in vitro and in vivo models are available for assessing theeffects of zacrp2 polypeptides, fragments, fusion proteins, antibodies,agonists and antagonists on ischemia and reperfusion injury. See forexample, Shandelya et al., Circulation 88:2812-26, 1993; Weisman et al.,Science 249:146-151, 1991; Buerke et al., Circulation 91:393-402, 1995;Horstick et al., Circulation 95:701-8, 1997 and Burke et al., J. Phar.Exp. Therp. 286:429-38, 1998. An ex vivo hamster platelet aggregationassay is described by Deckmyn et al., ibid. Bleeding times in hamstersand baboons can be measured following injection of zacrp2 polypeptidesusing the model described by Deckmyn et al., ibid. The formation ofthrombus in response to administration of proteins of the presentinvention can be measured using the hamster femoral vein thrombosismodel is provided by Deckmyn et al., ibid. Changes in platelet adhesionunder flow conditions following administration of zacrp2 can be measuredusing the method described in Harsfalvi et al., Blood 85:705-11, 1995.

Complement inhibition and wound healing can be zacrp2 polypeptides,fragments, fusion proteins, antibodies, agonists or antagonists beassayed alone or in combination with other know inhibitors ofcollagen-induced platelet activation and aggregation, such as palldipin,moubatin or calin, for example.

Zacrp2 polypeptides, fragments, fusion proteins, antibodies, agonists orantagonists can be evaluated using methods described herein or known inthe art, such as healing of dermal layers in pigs (Lynch et al., Proc.Natl. Acad. Sci. USA 84: 7696-700, 1987) and full-thickness skin woundsin genetically diabetic mice (Greenhalgh et al., Am. J. Pathol. 136:1235-46, 1990), for example. The polypeptides of the present inventioncan be assayed alone or in combination with other known complementinhibitors as described above.

Radiation hybrid mapping is a somatic cell genetic technique developedfor constructing high-resolution, contiguous maps of mammalianchromosomes (Cox et al., Science 250:245-50, 1990). Partial or fullknowledge of a gene's sequence allows the designing of PCR primerssuitable for use with chromosomal radiation hybrid mapping panels.Commercially available radiation hybrid mapping panels which cover theentire human genome, such as the Stanford G3 RH Panel and the GeneBridge4 RH Panel (Research Genetics, Inc., Huntsville, Ala.), are available.These panels enable rapid, PCR based, chromosomal localizations andordering of genes, sequence-tagged sites (STSs), and othernonpolymorphic- and polymorphic markers within a region of interest.This includes establishing directly proportional physical distancesbetween newly discovered genes of interest and previously mappedmarkers. The precise knowledge of a gene's position can be useful in anumber of ways including: 1) determining if a sequence is part of anexisting contig and obtaining additional surrounding genetic sequencesin various forms such as YAC-, BAC- or cDNA clones, 2) providing apossible candidate gene for an inheritable disease which shows linkageto the same chromosomal region, and 3) for cross-referencing modelorganisms such as mouse which may be beneficial in helping to determinewhat function a particular gene might have.

The results showed linkage of zacrp2 to the human chromosome 5 frameworkmarker SHGC-57747 with a LOD score of 7.80 and at a distance of 20cR_(—)10000 from the marker. The use of surrounding markers positionedzacrp2 in the 5q34 region on the integrated LDB human chromosome 5 map.The present invention also provides reagents which will find use indiagnostic applications. For example, the zacrp2 gene, a probecomprising zacrp2 DNA or RNA, or a subsequence thereof can be used todetermine if the zacrp2 gene is present on chromosome 5 or if a mutationhas occurred. Detectable chromosomal aberrations at the zacrp2 genelocus include, but are not limited to, aneuploidy, gene copy numberchanges, insertions, deletions, restriction site changes andrearrangements. These aberrations can occur within the coding sequence,within introns, or within flanking sequences, including upstreampromoter and regulatory regions, and may be manifested as physicalalterations within a coding sequence or changes in gene expressionlevel.

In general, these diagnostic methods comprise the steps of (a) obtaininga genetic sample from a patient; (b) incubating the genetic sample witha polynucleotide probe or primer as disclosed above, under conditionswherein the polynucleotide will hybridize to complementarypolynucleotide sequence, to produce a first reaction product; and (iii)comparing the first reaction product to a control reaction product. Adifference between the first reaction product and the control reactionproduct is indicative of a genetic abnormality in the patient. Geneticsamples for use within the present invention include genomic DNA, cDNA,and RNA. The polynucleotide probe or primer can be RNA or DNA, and willcomprise a portion of SEQ ID NO:1, the complement of SEQ ID NO:1, or anRNA equivalent thereof. Suitable assay methods in this regard includemolecular genetic techniques known to those in the art, such asrestriction fragment length polymorphism (RFLP) analysis, short tandemrepeat (STR) analysis employing PCR techniques, ligation chain reaction(Barany, PCR Methods and Applications 1:5-16, 1991), ribonucleaseprotection assays, and other genetic linkage analysis techniques knownin the art (Sambrook et al., ibid.; Ausubel et. al., ibid.; Marian,Chest 108:255-65, 1995). Ribonuclease protection assays (see, e.g.,Ausubel et al., ibid., ch. 4) comprise the hybridization of an RNA probeto a patient RNA sample, after which the reaction product (RNA-RNAhybrid) is exposed to RNase. Hybridized regions of the RNA are protectedfrom digestion. Within PCR assays, a patient's genetic sample isincubated with a pair of polynucleotide primers, and the region betweenthe primers is amplified and recovered. Changes in size or amount ofrecovered product are indicative of mutations in the patient. AnotherPCR-based technique that can be employed is single strand conformationalpolymorphism (SSCP) analysis (Hayashi, PCR Methods and Applications1:34-8, 1991).

Zacrp2 polypeptides may be used in the analysis of energy efficiency ofa mammal. Zacrp2 polypeptides found in serum or tissue samples may beindicative of a mammals ability to store food, with more highlyefficient mammals tending toward obesity. More specifically, the presentinvention contemplates methods for detecting zacrp2 polypeptidecomprising:

exposing a sample possibly containing zacrp2 polypeptide to an antibodyattached to a solid support, wherein said antibody binds to an epitopeof a zacrp2 polypeptide;

washing said immobilized antibody-polypeptide to remove unboundcontaminants;

exposing the immobilized antibody-polypeptide to a second antibodydirected to a second epitope of a zacrp2 polypeptide, wherein the secondantibody is associated with a detectable label; and

detecting the detectable label. The concentration of zacrp2 polypeptidein the test sample appears to be indicative of the energy efficiency ofa mammal. This information can aid nutritional analysis of a mammal.Potentially, this information may be useful in identifying and/ortargeting energy deficient tissue.

A further aspect of the invention provides a method for studyinginsulin. Such methods of the present invention comprise incubatingadipocytes in a culture medium comprising zacrp2 polypeptide, monoclonalantibody, agonist or antagonist thereof ± insulin and observing changesin adipocyte protein secretion or differentiation.

Anti-microbial protective agents may be directly acting or indirectlyacting. Such agents operating via membrane association or pore formingmechanisms of action directly attach to the offending microbe.Anti-microbial agents can also act via an enzymatic mechanism, breakingdown microbial protective substances or the cell wall/membrane thereof.Anti-microbial agents, capable of inhibiting microorganism proliferationor action or of disrupting microorganism integrity by either mechanismset forth above, are useful in methods for preventing contamination incell culture by microbes susceptible to that anti-microbial activity.Such techniques involve culturing cells in the presence of an effectiveamount of said zacrp2 polypeptide or an agonist or antagonist thereof.

Also, zacrp2 polypeptides or agonists thereof may be used as cellculture reagents in in vitro studies of exogenous microorganisminfection, such as bacterial, viral or fungal infection. Such moietiesmay also be used in in vivo animal models of infection.

The present invention also provides methods of studying mammaliancellular metabolism. Such methods of the present invention compriseincubating cells to be studied, for example, human vascular endothelialcells, ± zacrp2 polypeptide, monoclonal antibody, agonist or antagonistthereof and observing changes in adipogenesis, gluconeogenesis,glycogenolysis, lipogenesis, glucose uptake, or the like.

An additional aspect of the invention provides a method for studyingdimerization or oligomerization. Such methods of the present inventioncomprise incubating zacrp2 polypeptides or fragments or fusion proteinsthereof containing a collagen-like domain alone or in combination withother polypeptides bearing collagen-like domains and observing theassociations formed between the collagen like domains. Such associationsare indicated by HPLC, circular dichroism or the like.

Zacrp2 polypeptides, fragments, fusion proteins, antibodies, agonists orantagonists of the present invention can be used in methods forpromoting blood flow within the vasculature of a mammal by reducing thenumber of platelets that adhere and are activated and the size ofplatelet aggregates. Used to such an end, Zacrp2 can be administeredprior to, during or following an acute vascular injury in the mammal.Vascular injury may be due to vascular reconstruction, including but notlimited to, angioplasty, coronary artery bypass graft, microvascularrepair or anastomosis of a vascular graft. Also contemplated arevascular injuries due to trauma, stroke or aneurysm. In other preferredmethods the vascular injury is due to plaque rupture, degradation of thevasculature, complications associated with diabetes and atherosclerosis.Plaque rupture in the coronary artery induces heart attack and in thecerebral artery induces stroke. Use of zacrp2 polypeptides, fragments,fusion proteins, antibodies, agonists or antagonists in such methodswould also be useful for ameliorating whole system diseases of thevasculature associated with the immune system, such as disseminatedintravascular coagulation (DIC) and SIDs. Additionally the complementinhibiting activity would be useful for treating non-vasculature immunediseases such as arteriolosclerosis.

A correlation has been found between the presence of C1q in localizedischemic myocardium and the accumulation of leukocytes followingcoronary occlusion and reperfusion. Release of cellular componentsfollowing tissue damage triggers complement activation which results intoxic oxygen products that may be the primary cause of myocardial damage(Rossen et al., Circ. Res. 62:572-84, 1998 and Tenner, ibid.). Blockingthe complement pathway was found to protect ischemic myocardium fromreperfusion injury (Buerke et al., J. Pharm. Exp. Therp. 286:429-38,1998). Proteins having complement inhibition and C1q binding activitywould be useful for such purposes.

Collagen and C1q binding capabilities of adipocyte complement relatedprotein homologs such as zacrp2 would be useful to pacify damagedcollagenous tissues preventing platelet adhesion, activation oraggregation, and the activation of inflammatory processes which lead tothe release of toxic oxygen products. By rendering the exposed tissueinert towards such processes as complement activity, thrombotic activityand immune activation, reduces the injurious effects of ischemia andreperfusion. In particular, such injuries would include trauma injuryischemia, intestinal strangulation, and injury associated with pre- andpost-establishment of blood flow. Such polypeptides would be useful inthe treatment of cardiopulmonary bypass ischemia and recesitation,myocardial infarction and post trauma vasospasm, such as stroke orpercutanious transluminal angioplasty as well as accidental orsurgical-induced vascular trauma.

Additionally such collagen- and C1q-binding polypeptides would be usefulto pacify prosthetic biomaterials and surgical equipment to render thesurface of the materials inert towards complement activation, thromboticactivity or immune activation. Such materials include, but are notlimited to, collagen or collagen fragment-coated biomaterials,gelatin-coated biomaterials, fibrin-coated biomaterials,fibronectin-coated biomaterials, heparin-coated biomaterials, collagenand gel-coated stents, arterial grafts, synthetic heart valves,artificial organs or any prosthetic application exposed to blood thatwill bind zsig37 at greater than 1×10⁸. Coating such materials can bedone using methods known in the art, see for example, Rubens, U.S. Pat.No. 5,272,074.

Complement and C1q play a role in inflammation. The complementactivation is initiated by binding of C1q to immunoglobulins (Johnston,Pediatr. Infect. Dis. J. 12:933-41, 1993; Ward and Ghetie, Therap.Immunol. 2:77-94, 1995). Inhibitors of C1q and complement would beuseful as anti-inflammatory agents. Such application can be made toprevent infection. Additionally, such inhibitors can be administrated toan individual suffering from inflammation mediated by complementactivation and binding of immune complexes to C1q. Inhibitors of C1q andcomplement would be useful in methods of mediating wound repair,enhancing progression in wound healing by overcoming impaired woundhealing. Progression in wound healing would include, for example, suchelements as a reduction in inflammation, fibroblasts recruitment, woundretraction and reduction in infection.

Ability of tumor cells to bind to collagen may contribute to themetastasis of tumors. Inhibitors of collagen binding are also useful formediating the adhesive interactions and metastatic spread of tumors(Noeske-Jungbult et al., U.S. Pat. No. 5,723,312).

In addition, zacrp2 polypeptides, fragments, fusions agonists orantagonists thereof may be therapeutically useful for anti-microbialapplications. For example, complement component C1q plays a role in hostdefense against infectious agents, such as bacteria and viruses. C1q isknown to exhibit several specialized functions. For example, C1qtriggers the complement cascade via interaction with bound antibody orC-reactive protein (CRP). Also, C1q interacts directly with certainbacteria, RNA viruses, mycoplasma, uric acid crystals, the lipid Acomponent of bacterial endotoxin and membranes of certain intracellularorganelles. C1q binding to the C1q receptor is believed to promotephagocytosis. C1q also appears to enhance the antibody formation aspectof the host defense system. See, for example, Johnston, Pediatr. Infect.Dis. J. 12(11): 933-41, 1993. Thus, soluble C1q-like molecules may beuseful as anti-microbial agents, promoting lysis or phagocytosis ofinfectious agents.

The positively charged, extracellular, triple helix, collagenous domainsof C1q and macrophage scavenger receptor were determined to play a rolein ligand binding and were shown to have a broad binding specificity forpolyanions (Acton et al., J. Biol. Chem. 268:3530-37, 1993).Lysophospholipid growth factor (lysophosphatidic acid, LPA) and othermitogenic anions localize at the site of damaged tissues and assist inwound repair. LPA exerts many biological effects including activation ofplatelets and up-regulation of matrix assembly. It is thought that LPAsynergizes with other blood coagulation factors and mediates woundhealing.

The collagenous domains of proteins such as C1q and macrophage scavengerreceptor are know to bind acidic phospholipids such as LPA. Theinteraction of zacrp2 polypeptides, fragments, fusions, agonists orantagonists with mitogenic anions such as LPA can be determined usingassays known in the art, see for example, Acton et al., ibid. Inhibitionof inflammatory processes by polypeptides and antibodies of the presentinvention would also be useful in preventing infection at the woundsite.

For pharmaceutical use, the proteins of the present invention can beformulated with pharmaceutically acceptable carriers for parenteral,oral, nasal, rectal, topical, transdermal administration or the like,according to conventional methods. In a preferred embodimentadministration is made at or near the site of vascular injury. Ingeneral, pharmaceutical formulations will include a zacrp2 protein incombination with a pharmaceutically acceptable vehicle, such as saline,buffered saline, 5% dextrose in water or the like. Formulations mayfurther include one or more excipients, preservatives, solubilizers,buffering agents, albumin to prevent protein loss on vial surfaces, etc.Methods of formulation are well known in the art and are disclosed, forexample, in Remington: The Science and Practice of Pharmacy, Gennaro,ed., Mack Publishing Co., Easton Pa., 19^(th) ed., 1995. Therapeuticdoses will generally be determined by the clinician according toaccepted standards, taking into account the nature and severity of thecondition to be treated, patient traits, etc. Determination of dose iswithin the level of ordinary skill in the art.

As used herein a “pharmaceutically effective amount” of a zsig37polypeptide, fragment, fusion protein, agonist or antagonist is anamount sufficient to induce a desired biological result. The result canbe alleviation of the signs, symptoms, or causes of a disease, or anyother desired alteration of a biological system. For example, aneffective amount of a zacrp2 polypeptide is that which provides eithersubjective relief of symptoms or an objectively identifiable improvementas noted by the clinician or other qualified observer. Such an effectiveamount of a zacrp2 polypeptide would provide, for example, inhibition ofcollagen-activated platelet activation and the complement pathway,including C1q, increase localized blood flow within the vasculature of apatient and/or reduction in injurious effects of ischemia andreperfusion. Effective amounts of the zacrp2 polypeptides can varywidely depending on the disease or symptom to be treated. The amount ofthe polypeptide to be administered and its concentration in theformulations, depends upon the vehicle selected, route ofadministration, the potency of the particular polypeptide, the clinicalcondition of the patient, the side effects and the stability of thecompound in the formulation. Thus, the clinician will employ theappropriate preparation containing the appropriate concentration in theformulation, as well as the amount of formulation administered,depending upon clinical experience with the patient in question or withsimilar patients. Such amounts will depend, in part, on the particularcondition to be treated, age, weight, and general health of the patient,and other factors evident to those skilled in the art. Typically a dosewill be in the range of 0.01-100 mg/kg of subject. In applications suchas balloon catheters the typical dose range would be 0.05-5 mg/kg ofsubject. Doses for specific compounds may be determined from in vitro orex vivo studies in combination with studies on experimental animals.Concentrations of compounds found to be effective in vitro or ex vivoprovide guidance for animal studies, wherein doses are calculated toprovide similar concentrations at the site of action.

Polynucleotides encoding zacrp2 polypeptides are useful within genetherapy applications where it is desired to increase or inhibit zacrp2activity. If a mammal has a mutated or absent zacrp2 gene, the zacrp2gene can be introduced into the cells of the mammal. In one embodiment,a gene encoding a zacrp2 polypeptide is introduced in vivo in a viralvector. Such vectors include an attenuated or defective DNA virus, suchas, but not limited to, herpes simplex virus (HSV), papillomavirus,Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV), andthe like. Defective viruses, which entirely or almost entirely lackviral genes, are preferred. A defective virus is not infective afterintroduction into a cell. Use of defective viral vectors allows foradministration to cells in a specific, localized area, without concernthat the vector can infect other cells. Examples of particular vectorsinclude, but are not limited to, a defective herpes simplex virus 1(HSV1) vector (Kaplitt et al., Molec. Cell. Neurosci. 2:320-30, 1991);an attenuated adenovirus vector, such as the vector described byStratford-Perricaudet et al., J. Clin. Invest. 90:626-30, 1992; and adefective adeno-associated virus vector (Samulski et al., J. Virol.61:3096-101, 1987; Samulski et al., J. Virol. 63:3822-8, 1989).

In another embodiment, a zacrp2 gene can be introduced in a retroviralvector, e.g., as described in Anderson et al., U.S. Pat. No. 5,399,346;Mann et al. Cell 33:153, 1983; Temin et al., U.S. Pat. No. 4,650,764;Temin et al., U.S. Pat. No. 4,980,289; Markowitz et al., J. Virol.62:1120, 1988; Temin et al., U.S. Pat. No. 5,124,263; WIPO PublicationWO 95/07358; and Kuo et al., Blood 82:845, 1993. Alternatively, thevector can be introduced by lipofection in vivo using liposomes.Synthetic cationic lipids can be used to prepare liposomes for in vivotransfection of a gene encoding a marker (Felgner et al., Proc. Natl.Acad. Sci. USA 84:7413-7, 1987; Mackey et al., Proc. Natl. Acad. Sci.USA 85:8027-31, 1988). The use of lipofection to introduce exogenousgenes into specific organs in vivo has certain practical advantages.Molecular targeting of liposomes to specific cells represents one areaof benefit. More particularly, directing transfection to particularcells represents one area of benefit. For instance, directingtransfection to particular cell types would be particularly advantageousin a tissue with cellular heterogeneity, such as the pancreas, liver,kidney, and brain. Lipids may be chemically coupled to other moleculesfor the purpose of targeting. Targeted peptides (e.g., hormones orneurotransmitters), proteins such as antibodies, or non-peptidemolecules can be coupled to liposomes chemically.

It is possible to remove the target cells from the body; to introducethe vector as a naked DNA plasmid; and then to re-implant thetransformed cells into the body. Naked DNA vectors for gene therapy canbe introduced into the desired host cells by methods known in the art,e.g., transfection, electroporation, microinjection, transduction, cellfusion, DEAE dextran, calcium phosphate precipitation, use of a gene gunor use of a DNA vector transporter. See, e.g., Wu et al., J. Biol. Chem.267:963-7, 1992; Wu et al., J. Biol. Chem. 263:14621-4, 1988.

Antisense methodology can be used to inhibit zacrp2 gene transcription,such as to inhibit cell proliferation in vivo. Polynucleotides that arecomplementary to a segment of a zacrp2-encoding polynucleotide (e.g., apolynucleotide as set froth in SEQ ID NO:1) are designed to bind tozacrp2-encoding mRNA and to inhibit translation of such mRNA. Suchantisense polynucleotides are used to inhibit expression of zacrp2polypeptide-encoding genes in cell culture or in a subject.

Transgenic mice, engineered to express the zacrp2 gene, and mice thatexhibit a complete absence of zacrp2 gene function, referred to as“knockout mice” (Snouwaert et al., Science 257:1083, 1992), may also begenerated (Lowell et al., Nature 366:740-42, 1993). These mice may beemployed to study the zacrp2 gene and the protein encoded thereby in anin vivo system.

The invention is further illustrated by the following non-limitingexamples.

EXAMPLE 1 Tissue Distribution

Northerns were performed using Human Multiple Tissue Blots (MTN1, MTN2and MTN3) from Clontech (Palo Alto, Calif.) were probed to determine thetissue distribution of human zacrp2. A 1039 bp cDNA probe correspondingto full length zacrp2 was obtained from the clone discussed above byrestriction digest with Eco RI and Hind III restriction enzymesaccording to manufacturer's instructions. The restriction fragment wasisolated by gel electrophoresis and purified using a Gel Extraction Kit(Qiagen, Chatsworth, Calif.) according to manufacturer's instructions.The probe was radioactively labeled using a Rediprime II DNA labelingkit (Amersham, Arlington Heights, Ill.) according to the manufacturer'sspecifications. The probe was purified using a NUCTRAP push column(Stratagene Cloning Systems, La Jolla, Calif.). EXPRESSHYB (Clontech,Palo Alto, Calif.) solution was used for prehybridization and as ahybridizing solution for the Northern blots. Hybridization took placeovernight at 55° C., using 1.5×10⁶ cpm/ml labeled probe. The blots werethen washed in 2×SSC and 0.1% SDS at room temperature, then with 2×SSCand 0.1% SDS at 65° C., followed by a wash in 0.1×SSC and 0.1% SDS at65° C. A single transcript of approximately 1.2 kb was seen in prostate,testis, liver, heart, stomach, thyroid, spinal cord and trachea.

A RNA Master Dot Blot (Clontech) that contained RNAs from varioustissues that were normalized to 8 housekeeping genes were also probedand hybridized as described above. Expression was seen at low levels inall tissues, with higher expression in heart, aorta, uterus, thyroid andsmall intestine.

EXAMPLE 2 Chromosomal Assignment and Placement of Zacrp2

Zacrp2 was mapped to human chromosome 5 using the commercially availableversion of the “Stanford G3 Radiation Hybrid Mapping Panel” (ResearchGenetics, Inc., Huntsville, Ala.). The Stanford G3 RH Panel containsPCRable DNAs from each of 83 radiation hybrid clones of the whole humangenome, plus two control DNAs (the RM donor and the A3 recipient). Apublicly available WWW server by Stanford University, which is locatedon the Internet, allows chromosomal localization of markers.

For the mapping of zacrp2 with the Stanford G3 RH Panel, 20 μl reactionswere set up in a 96-well microtiter plate (Stratagene, La Jolla, Calif.)and used in a RoboCycler Gradient 96 thermal cycler (Stratagene). Eachof the 85 PCR reactions consisted of 2 μl 10× KlenTaq PCR reactionbuffer (Clontech), 1.6 μl dNTPs mix (2.5 mM each, PERKIN-ELMER, FosterCity, Calif.), 1 μl sense primer, ZC 20,810 (SEQ ID NO:13), 1 μlantisense primer, ZC 20,809 (SEQ ID NO:14), 2 μl RediLoad (ResearchGenetics, Inc.), 0.4 μl 50× Advantage KlenTaq Polymerase Mix (ClontechLaboratories, Inc.), 25 ng of DNA from an individual hybrid clone orcontrol and ddH₂O for a total volume of 20 μl. The reactions wereoverlaid with an equal amount of mineral oil and sealed. The PCR cyclerconditions were as follows: an initial 1 cycle 5 minute denaturation at94° C., 35 cycles of a 45 seconds denaturation at 94° C., 45 secondsannealing at 62° C. and 1 minute AND 15 seconds extension at 72° C.,followed by a final 1 cycle extension of 7 minutes at 72° C. Thereactions were separated by electrophoresis on a 2% agarose gel (LifeTechnologies, Gaithersburg, Md.).

The results showed linkage of zacrp2 to the human chromosome 5 frameworkmarker SHGC-57747 with a LOD score of 7.80 and at a distance of 20cR_(—)10000 from the marker. The use of surrounding markers positionszacrp2 in the 5q34 region on the integrated LDB human chromosome 5 map(The Genetic Location Database, University of Southhampton).

EXAMPLE 3 Baculovirus Expression of Human zacrp2

An expression vector, pzacrp2cee, was prepared to express human zacrp2polypeptides having a C-terminal Glu-Glu tag, in insect cells.

A. Construction of pzacrp2cee

An 887 bp fragment containing sequence encoding zacrp2 andpolynucleotide sequence encoding BamHI and Xba1 restriction sites on the5′ and 3′ ends, respectively, was generated by PCR amplification from aplasmid containing zacrp2 cDNA using primers ZC23375 (SEQ ID NO:15) andZC23376 (SEQ ID NO:16). The PCR reaction conditions were as follows: 1cycle of 94° C. for 4 minutes, followed by 25 cycles of 94° C. for 45seconds, 50° C. for 45 seconds, and 72° C. for 2 minutes; 1 cycle at 72°C. for 10 min; followed by a 4° C. soak. The fragment was visualized bygel electrophoresis (1% SeaPlaque/1% NuSieve). The band was excised,diluted to 0.5% agarose with 2 mM MgCl₂, melted at 65° C. and ligatedinto an BamHI/XbaI digested baculovirus expression donor vector,pZBV32L. The pZBV32L vector is a modification of the pFastBac1™ (LifeTechnologies, Grand Island, N.Y.) expression vector, where thepolyhedron promoter has been removed and replaced with the lateactivating Basic Protein Promoter and the coding sequence for theGlu-Glu tag (SEQ ID NO:17) as well as a stop signal is inserted at the3′ end of the multiple cloning region). About 28 nanograms of therestriction digested zacrp2 insert and about 56 ng of the correspondingvector were ligated overnight at 16° C. The ligation mix was diluted 3fold in TE (10 mM Tris-HCl, pH 7.5 and 1 mM EDTA) and 4 fmol of thediluted ligation mix was transformed into DH5α Library Efficiencycompetent cells (Life Technologies) according to manufacturer'sdirection by heat shock for 45 seconds in a 42° C. waterbath. Thetransformed DNA and cells were diluted in 450 μl of SOC media (2% BactoTryptone, 0.5% Bacto Yeast Extract, 10 ml 1M NaCl, 1.5 mM KCl, 10 mMMgCl₂, 10 mM MgSO₄ and 20 mM glucose) and plated onto LB platescontaining 100 μg/ml ampicillin. Clones were analyzed by restrictiondigests and 1 μl of the positive clone was transformed into 20 μlDH10Bac Max Efficiency competent cells (GIBCO-BRL, Gaithersburg, Md.)according to manufacturer's instruction, by heat shock for 45 seconds ina 42° C. waterbath. The transformed cells were then diluted in 980 μlSOC media (2% Bacto Tryptone, 0.5% Bacto Yeast Extract, 10 ml 1M NaCl,1.5 mM KCl, 10 mM MgCl₂, 10 mM MgSO₄ and 20 mM glucose) out grown inshaking incubator at 37° C. for four hours and plated onto Luria Agarplates containing 50 μg/ml kanamycin, 7 μg/ml gentamicin, 10 μg/mltetracycline, IPTG and Bluo Gal. The plated cells were incubated for 48hours at 37° C. A color selection was used to identify those cellshaving zacrp2cee encoding donor insert that had incorporated into theplasmid (referred to as a “bacmid”). Those colonies, which were white incolor, were picked for analysis. Bacmid DNA was isolated from positivecolonies using the QiaVac Miniprep8 system (Qiagen) according themanufacturer's directions. Clones were screened for the correct insertby amplifying DNA using primers to the transposable element in thebacmid via PCR using primers ZC447 (SEQ ID NO:18) and ZC976 (SEQ IDNO:19). The PCR reaction conditions were as follows: 35 cycles of 94° C.for 45 seconds, 50° C. for 45 seconds, and 72° C. for 5 minutes; 1 cycleat 72° C. for 10 min.; followed by 4° C. soak. The PCR product was runon a 1% agarose gel to check the insert size. Those having the correctinsert were used to transfect Spodoptera frugiperda (Sf9) cells.

B. Transfection

Sf9 cells were seeded at 5×10⁶ cells per 35 mm plate and allowed toattach for 1 hour at 27° C. Five microliters of bacmid DNA was dilutedwith 100 μl Sf-900 II SFM (Life Technologies). Six μl of CellFECTINReagent (Life Technologies) was diluted with 100 μl Sf-900 II SFM. Thebacmid DNA and lipid solutions were gently mixed and incubated 30-45minutes at room temperature. The media from one plate of cells wereaspirated, the cells were washed 1× with 2 ml fresh Sf-900 II SFM media.Eight hundred microliters of Sf-900 II SFM was added to the lipid-DNAmixture. The wash media was aspirated and the DNA-lipid mix added to thecells. The cells were incubated at 27° C. for 4-5 hours. The DNA-lipidmix was aspirated and 2 ml of Sf-900 II media was added to each plate.The plates were incubated at 27° C., 90% humidity, for 96 hours afterwhich the virus was harvested.

C. Primary Amplification

Sf9 cells were grown in 50 ml Sf-900 II SFM in a 125 ml shake flask toan approximate density of 0.41-0.52×10⁵ cells/ml. They were theninfected with 150 μl of the virus stock from above and incubated at 27°C. for 3 days after which time the virus was harvested according tostandard methods known in the art.

EXAMPLE 4 Purification of Baculovirus Expressed Glu-Glu-Tagged zacrp2Polypeptides

Unless otherwise noted, all operations were carried out at 4° C. Amixture of protease inhibitors were added to a 2 liter sample ofconditioned media from C-terminal Glu-Glu (EE) tagged zacrp2baculovirus-infected Sf9 cells to final concentrations of 2.5 mMethylenediaminetetraacetic acid (EDTA, Sigma Chemical Co. St. Louis,Mo.), 0.001 mM leupeptin (Boehringer-Mannheim, Indianapolis, Ind.),0.001 mM pepstatin (Boehringer-Mannheim) and 0.4 mM Pefabloc(Boehringer-Mannheim). The sample was centrifuged at 10,000 rpm for 30min at 4° C. in a Beckman JLA-10.5 rotor (Beckman Instruments) in aBeckman Avanti J25I centrifuge (Beckman Instruments) to remove celldebris. To the supernatant fraction was added a 50.0 ml sample ofanti-EE Sepharose, prepared as described below, and the mixture wasgently agitated on a Wheaton (Millville, N.J.) roller culture apparatusfor 18.0 h at 4° C.

The mixture was poured into a 5.0×20.0 cm Econo-Column (Bio-RadLaboratories) and the gel was washed with 30 column volumes of phosphatebuffered saline (PBS). The unretained flow-through fraction wasdiscarded. Once the absorbance of the effluent at 280 nM was less than0.05, flow through the column was reduced to zero and the anti-EESepharose gel was washed with 2.0 column volumes of PBS containing 0.2mg/ml of EE peptide (AnaSpec, San Jose, Calif.). The peptide used hasthe sequence Glu-Tyr-Met-Pro-Val-Asp (SEQ ID NO:20). After 1.0 hour at4° C., flow was resumed and the eluted protein was collected. Thisfraction was referred to as the peptide elution. The anti-EE Sepharosegel was washed with 2.0 column volumes of 0.1 M glycine, pH 2.5, and theglycine wash was collected separately. The pH of the glycine-elutedfraction was adjusted to 7.0 by the addition of a small volume of 10×PBSand stored at 4° C.

The peptide elution was concentrated to 5.0 ml using a 5,000 molecularweight cutoff membrane concentrator (Millipore) according to themanufacturer's instructions. The concentrated peptide elution wasseparated from free peptide by chromatography on a 1.5×50 cm SephadexG-50 (Pharmacia) column equilibrated in PBS at a flow rate of 1.0 ml/minusing a BioCad Sprint HPLC (PerSeptive BioSystems). Two ml fractionswere collected and the absorbance at 280 nM was monitored. The firstpeak of material absorbing at 280 nM and eluting near the void volume ofthe column was collected. This material represented purified zacrp2CEE.These bands were present in about equimolar amounts. Both bands showedcross-reactivity with anti-EE antibodies by Western blotting of thepurified material. The protein concentration (0.254 mg/ml) of thepurified proteins was determined by BCA analysis (Pierce) and thematerial was aliquoted, and stored at −80° C.

Preparation of Anti-EE Sepharose

A 100 ml bed volume of protein G-Sepharose (Pharmacia) was washed 3times with 100 ml of PBS containing 0.02% sodium azide using a 500 mlNalgene 0.45 micron filter unit. The gel was washed with 6.0 volumes of200 mM triethanolamine, pH 8.2 (TEA, Sigma), and an equal volume of EEantibody solution containing 900 mg of antibody was added. After anovernight incubation at 4° C., unbound antibody was removed by washingthe resin with 5 volumes of 200 mM TEA as described above. The resin wasresuspended in 2 volumes of TEA, transferred to a suitable container,and dimethylpimilimidate-2 HCl (Pierce), dissolved in TEA, was added toa final concentration of 36 mg/ml of gel. The gel was rocked at roomtemperature for 45 min and the liquid was removed using the filter unitas described above. Nonspecific sites on the gel were then blocked byincubating for 10 minutes at room temperature with 5 volumes of 20 mMethanolamine in 200 mM TEA. The gel was then washed with 5 volumes ofPBS containing 0.02% sodium azide and stored in this solution at 4° C.

EXAMPLE 5 Adhesion Molecule Assays

Upon stimulation with inflammatory cytokines such as TNF (tumor necrosisfactor), human microvascular bone marrow cells (TRBMEC) express cellsurface adhesion molecules, including E-selectin (endothelial leukocyteadhesion molecule), V-CAM (vascular cell adhesion molecule), and I-CAM(intercellular adhesion molecule).

The effect of zacrp2 on expression of cell surface adhesion moleculeswas determined using microvascular bone marrow cells (TRBMEC) in a cellELISA according to Ouchi et al., (Circulation 100:2473-7, 1999).Briefly, TRBMEC cells were grown in 96 well, flat bottom plates (Costar,Pleasanton, Calif.) until confluent. Both wild type control media andbaculovirus-expressed zacrp2 media were 10× before testing (CentriconCentrifugal Filtration Unit 5,000K cutoff, Millipore Corp., Bedford,Mass.) according to manufacturer's instructions. To each well 90 μl ofzacrp2-containing media or control media was added, and the plates wereincubated at 37° C., 5% CO₂ overnight. The next day, half of the samplesreceived 10 μl of TNFα (10 ng/ml, R&D Systems, Minneapolis, Minn.), theother samples were untreated, measuring basal expression. The plateswere incubated at 37° C., 5% CO₂ for 4 hours.

Following incubation, the media was removed from the plates and 50 μlanti-human VCAM antibody (1:1000 dilution of 1 mg/ml stock, R&DSystems), 50 μl of anti-human ICAM-1 monoclonal antibody (1:1000dilution of a 1 mg/ml stock, R&D Systems), or 50 μl of anti-humanE-selectin antibodies (1:1000 dilution of a 1 mg/ml stock, R&D Systems)were then added to triplicate wells and the plates were incubated at 37°C., 5% CO₂ for 1 hour.

The antibody solution was removed and the plates were washed three timesin warm RPMI+5% FBS. Following the last wash, 100 μl/well of an 0.05%gluteraldehyde solution (1:1000 of 50% gluteraldehyde in PBS) was addedto the wells and the plates were incubated at room temperature for 10minutes. The plates were washed three times with PBS and 50 μl/well ofsecondary antibody (1:1000 dilution of goat α-mouse IgG whole moleculeHRP conjugate (Sigma Chemical Co., St. Louis, Mo.) was added to allwells. The plates were incubated for one hour at 37° C.

The plates were then washed five times with washing buffer (PBS+0.05%Tween 20) and 100 μl/well TMB solution (100 μl of 4 mg/ml Tetra methylbenzidine (Sigma) in DMSO, in 10 ml 60 mM Na Acetate pH 5.0 and 100 μl1.2% H₂O₂) was added to each well. The plates were allowed to develop atroom temperature for 15-20 minutes at which time the reaction wasquenched by adding 100 μl/well 1M H₂SO₄. Plates were read at 450 nm withreference wavelength of 655 nm.

Zacrp2 showed no effect on ICAM-1 expression. Zacrp2 did have an effecton VCAM-1 expression. When compared to the maximal TNF response, zacrp2treated cells showed about 50% inhibition. Zacrp2 also had an effect,although less, about 10% inhibition on E-selection expression.

VCAM-1 expression was measured following direct adenovirus infection ofTRBMEC cells. Briefly, TRBMEC cells were directly infected with anadenovirus containing zacrp2 or the parental adenovirus strain. Thevirus was added at various multiplicities of infection (moi 500, 1,000and 5,000). Cells were incubated at 37° C., 5% CO₂ for 43 hours.Following infection, the adenovirus-infected cells were challenged withTNFα (1 ng/ml) for 4 hours. VCAM expression was measured as describedabove. VCAM-1 expression was dose dependent, with greatest inhibition,20%, at a multiplicity of infection of 5000.

A THP-1 monocyte adherence assay according to Ouchi et al., (ibid.) andCybulsky and Gimbrone, (Science 251:788-91, 1991) showed the sameresults as were seen for VCAM-1 above.

1. An isolated polynucleotide encoding a polypeptide wherein the encodedpolypeptide comprises a sequence of amino acid residues that is at least95% identical in amino acid sequence to residues 16-285 of SEQ ID NO:2,and wherein said sequence comprises: Gly-Xaa-Xaa or Gly-Xaa-Pro repeatsforming a collagen domain, wherein Xaa is any amino acid; and acarboxyl-terminal C1q domain comprising 10 beta strands.
 2. The isolatedpolynucleotide of claim 1, wherein the encoded polypeptide is at least95% identical in amino acid sequence to residues 1-285 of SEQ ID NO:2.3. The isolated polynucleotide of claim 1, wherein said collagen domainconsists of 24 Gly-Xaa-Xaa repeats and 10 Gly-Xaa-Pro repeats.
 4. Theisolated polynucleotide of claim 1, wherein said carboxyl-terminal C1qdomain comprises the sequence of SEQ ID NO:5.
 5. The isolatedpolynucleotide of claim 1, wherein said carboxy-terminal C1q domaincomprises amino acid residues 151-155, 172-174, 180-183, 187-190,193-205, 208-214, 220-227, 229-241, 246-251 and 269-274 of SEQ ID NO:2.6. The isolated polynucleotide of claim 1, wherein any differencesbetween said polypeptide and SEQ ID NO:2 are due to conservative aminoacid substitutions.
 7. The isolated polynucleotide of claim 1, whereinsaid polypeptide specifically binds with an antibody that specificallybinds with a polypeptide consisting of the amino acid sequence of SEQ IDNO:2.
 8. The isolated polynucleotide of claim 1, wherein said collagendomain consists of amino acid residues 41-141 of SEQ ID NO:2.
 9. Theisolated polynucleotide of claim 1, wherein said carboxy-terminal C1qdomain consists of amino acid residues 142-285 of SEQ ID NO:2.
 10. Theisolated polynucleotide of claim 1, wherein said polypeptide comprisesresidues 16-285 of SEQ ID NO:2.
 11. An isolated polynucleotidecomprising a sequence selected from the group consisting of: a)nucleotide 1 to nucleotide 1161 of SEQ ID NO:1; b) nucleotide 133 tonucleotide 987 of SEQ ID NO:1; c) nucleotide 133 to nucleotide 990 ofSEQ ID NO:1; d) nucleotide 178 to nucleotide 987 of SEQ ID NO:1; e)nucleotide 178 to nucleotide 990 of SEQ ID NO:1; f) nucleotide sequencescomplementary to a), b), c), d), or e).
 12. An isolated polynucleotideencoding a fusion protein, wherein the fusion protein comprises a firstportion and a second portion joined by a peptide bond, and wherein thefirst portion comprises a polypeptide comprising amino acid residues16-285 of SEQ ID NO:2, and wherein the second portion comprises anotherpolypeptide.
 13. An expression vector comprising the following operablylinked elements: a transcription promoter; an isolated DNA segmentencoding a polypeptide comprising a sequence of amino acid residues thatis at least 95% identical in amino acid sequence to residues 16-285 ofSEQ ID NO:2, wherein said sequence comprises: Gly-Xaa-Xaa or Gly-Xaa-Prorepeats forming a collagen domain, wherein Xaa is any amino acid; and acarboxyl-terminal C1q domain comprising 10 beta strands; and atranscription terminator.
 14. An expression vector of claim 13, whereinsaid DNA segment encodes a polypeptide that is at least 95% identical inamino acid sequence to residues 1-285 of SEQ ID NO:2.
 15. The expressionvector of claim 13, wherein said collagen domain consists of 24Gly-Xaa-Xaa repeats and 10 Gly-Xaa-Pro repeats.
 16. The expressionvector of claim 13, wherein said carboxyl-terminal C1q domain comprisesa sequence of SEQ ID NO:5.
 17. The expression vector of claim 13,wherein said carboxy-terminal C1q domain comprises amino acid residues151-155, 172-174, 180-183, 187-190, 193-205, 208-214, 220-227, 229-241,246-251 and 269-274 of SEQ ID NO:2.
 18. The expression vector of claim13, wherein any differences between said polypeptide and SEQ ID NO:2 aredue to conservative amino acid substitutions.
 19. The expression vectorof claim 13, wherein said polypeptide specifically binds with anantibody that specifically binds with a polypeptide consisting of theamino acid sequence of SEQ ID NO:2.
 20. The expression vector of claim13, wherein said collagen domain consists of amino acid residues 41-141of SEQ ID NO:2.
 21. The expression vector of claim 13, wherein saidcarboxy-terminal C1q domain consists of amino acid residues 142-285 ofSEQ ID NO:2.
 22. The expression vector of claim 13, wherein said DNAsegment encodes a polypeptide comprising amino acid residues 16-285 ofSEQ ID NO:2.
 23. The expression vector of claim 13, wherein said DNAsegment encodes a polypeptide covalently linked at the amino or carboxylterminus to an affinity tag.
 24. The expression vector of claim 13,wherein said DNA segment further encodes a secretory signal sequenceoperably linked to said polypeptide.
 25. The expression vector of claim13, wherein said secretory signal sequence comprises residues 1-15 ofSEQ ID NO:2.
 26. A cultured cell into which has been introduced anexpression vector according to claim 13, wherein said cell expressessaid polypeptide encoded by said DNA segment.
 27. A method of producinga polypeptide comprising: culturing a cell into which has beenintroduced an expression vector according to claim 13; whereby said cellexpresses said polypeptide encoded by said DNA segment; and recoveringsaid expressed polypeptide.